WO2019131978A1 - Scaffolding material for stem cell cultures and stem cell culture method using same - Google Patents

Scaffolding material for stem cell cultures and stem cell culture method using same Download PDF

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Publication number
WO2019131978A1
WO2019131978A1 PCT/JP2018/048386 JP2018048386W WO2019131978A1 WO 2019131978 A1 WO2019131978 A1 WO 2019131978A1 JP 2018048386 W JP2018048386 W JP 2018048386W WO 2019131978 A1 WO2019131978 A1 WO 2019131978A1
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WIPO (PCT)
Prior art keywords
stem cell
cell culture
group
acid
polyvinyl acetal
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PCT/JP2018/048386
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French (fr)
Japanese (ja)
Inventor
聡 羽根田
百里子 真鍋
亮馬 石井
博貴 井口
山内　博史
大村　貴宏
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積水化学工業株式会社
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Application filed by 積水化学工業株式会社 filed Critical 積水化学工業株式会社
Priority to EP23203425.6A priority Critical patent/EP4286508A3/en
Priority to ES18893713T priority patent/ES2966599T3/en
Priority to US16/958,204 priority patent/US20200362289A1/en
Priority to KR1020207021349A priority patent/KR102370115B1/en
Priority to JP2019562491A priority patent/JP6789416B2/en
Priority to CN201880083523.3A priority patent/CN111511896A/en
Priority to AU2018398050A priority patent/AU2018398050B2/en
Priority to EP18893713.0A priority patent/EP3733834B1/en
Priority to SG11202005433SA priority patent/SG11202005433SA/en
Publication of WO2019131978A1 publication Critical patent/WO2019131978A1/en
Priority to US16/919,312 priority patent/US20200407672A1/en

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    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
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    • C08L79/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen or carbon only, not provided for in groups C08L61/00 - C08L77/00
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    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C08L2666/00—Composition of polymers characterized by a further compound in the blend, being organic macromolecular compounds, natural resins, waxes or and bituminous materials, non-macromolecular organic substances, inorganic substances or characterized by their function in the composition
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides

Definitions

  • the present invention relates to a scaffold material for stem cell culture and a stem cell culture method using the same.
  • Stem cells are expected to be applied to drug discovery and regenerative medicine.
  • Stem cells are cells having self-replication ability and differentiation ability, and pluripotent stem cells capable of differentiating into all cell types, and tissue stem cells and tissue precursor cells capable of differentiating only into constituent cell types of somatic body tissues. is there.
  • pluripotent stem cells include human embryonic stem cells (hESCs) and human pluripotent stem cells (hPSCs) such as human induced pluripotent stem cells (hiPSCs). Culturing and expanding stem cells safely and reproducibly is an essential basic technology for medical application of these cells. In particular, in industrial use of regenerative medicine, it is necessary to treat stem cells in a large amount in an undifferentiated state.
  • a stem cell culture resin carrier using synthetic resin is proposed.
  • a polyvinyl acetal compound having a degree of acetalization of 20 to 60 mol% to provide a scaffold having excellent hydrophilicity and water resistance in culturing mouse fibroblasts. It is done.
  • the column of Example of Patent Document 2 discloses a hydrogel composed of an acrylic polymer in the culture of mouse ES cells.
  • the column of the example of Patent Document 3 discloses a hydrophilic and flexible polyrotaxane gel in the culture of mouse iPS cells.
  • Patent Document 1 has a problem that it swells in a culture medium due to its high hydrophilicity and the scaffold resin is peeled off. In addition, there is a problem that the fixation after seeding of stem cells and pluripotent stem cells is low, and the cells do not proliferate sufficiently.
  • Patent Document 2 uses sodium salt of 2-acrylamido-2-methylpropane sulfonic acid and sodium p-styrene sulfonate, N, N'-dimethyl acrylamide, and has high hydrophilicity and thus swells in a medium. And there is a problem that the scaffold resin is peeled off.
  • Patent Document 3 has a problem that it swells in a culture medium due to its high hydrophilicity and the scaffold resin is peeled off. In addition, there is a problem that the differentiation to cardiomyocytes is promoted because it is a flexible scaffold.
  • An object of the present invention is to provide a scaffold material for stem cell culture which has appropriate hydrophilicity and strength, is highly stable after seeding of stem cells, and can efficiently proliferate cells, and a stem cell culture method using the same. I assume.
  • the present invention relates to the following contents.
  • a scaffold material for stem cell culture which has a dispersion component ⁇ d of surface free energy of 24.5 or more and less than 45.0 and a dipole component ⁇ p of surface free energy of 1.0 or more and less than 20.0.
  • the scaffold material for stem cell culture according to (1), wherein the scaffold material for stem cell culture comprises a synthetic resin.
  • the synthetic resin contains at least one of a polyvinyl acetal skeleton and a poly (meth) acrylic acid ester skeleton.
  • the polyvinyl acetal resin is such that the total content of the structural unit having an imine structure, the structural unit having an amino group, and the structural unit having an amide structure is 0.1 mol% or more and 20 mol% or less 6)
  • a container for stem cell culture comprising a resin membrane comprising the scaffold material for stem cell culture according to any one of (1) to (8) in at least a part of a cell culture region.
  • a stem cell culture fiber comprising the scaffold material for stem cell culture according to any one of (1) to (8).
  • (11) A method for culturing stem cells using the scaffold material according to any one of (1) to (8).
  • a scaffold material for stem cell culture which has appropriate hydrophilicity and strength, and which has high fixation after seeding of stem cells, and a stem cell culture method using the same.
  • FIG. 1 is a diagram summarizing the relationship of ⁇ p to ⁇ d of the main synthetic resin.
  • FIG. 2 is a partially enlarged view of FIG.
  • FIG. 3 is a partially enlarged view of FIG.
  • FIG. 4 is a diagram showing the evaluation criteria of initial adhesion 24 hours after cell seeding.
  • FIG. 5 is a phase contrast micrograph of a scaffold for stem cell culture according to an example 24 hours after seeding of cells.
  • FIG. 6 is a phase contrast micrograph of a scaffold for stem cell culture according to a comparative example 24 hours after seeding of cells.
  • FIG. 7 shows the evaluation criteria of cell proliferation 5 days after cell seeding.
  • FIG. 8 is a phase contrast photomicrograph five days after seeding of cells in the scaffold for stem cell culture according to the example.
  • FIG. 9 is a phase contrast micrograph of a scaffold for stem cell culture according to a comparative example, five days after seeding of cells.
  • stem cells refer to cells having self-replication ability and differentiation ability.
  • stem cells those capable of self-replication and capable of differentiating from one cell to all cells of endoderm, mesoderm and ectoderm are referred to as "pluripotent stem cells”.
  • pluripotent stem cells for example, induced pluripotent stem cells (hereinafter referred to as "iPS cells"), embryonic stem cells (hereinafter referred to as “ES cells”), Muse cells (multilinege) Differentiating stress enduring cells), embryonic cancer cells (embryonic germ cells), embryonic germ stem cells (embryonic germ cells), mGS cells (multipotent germ stem cells) and the like can be mentioned.
  • iPS cells induced pluripotent stem cells
  • ES cells embryonic stem cells
  • Muse cells multilinege
  • embryonic cancer cells embryonic germ cells
  • embryonic germ stem cells embryonic germ stem cells
  • mGS cells multipotent germ stem cells
  • Tissue stem cells and tissue precursor cells include, for example, neural stem cells, neural crest stem cells, retinal stem cells, corneal stem cells, keratinocyte epidermal stem cells, melanocyte stem cells, mammary stem cells, hepatic stem cells, enteric stem cells, airway stem cells, hematopoietic stem cells, mesenchymal stem cells Cardiac stem cells, vascular endothelial progenitor cells, pericytes of vascular blood, skeletal muscle stem cells, adipose stem cells, renal progenitor cells, sperm stem cells and the like.
  • stem cells there can be mentioned, for example, the stem cells described in “More clearly! Stem cells and regenerative medicine” (by Yodosha, Kenji Nagafune).
  • the present inventors find out that the above-mentioned subject can be solved by controlling the surface free energy of the scaffold material for stem cell culture, and came to complete the present invention. That is, the first aspect of the present invention, the dispersion component gamma d and the dipole component gamma p of the surface free energy, to scaffolds for stem cell culture in a certain range.
  • the dispersion component gamma d and the dipole component gamma p of the surface free energy in this specification can be measured using the Kaelble-Uy theoretical formula.
  • Kaelble-Uy as represented by the formula (1), based on the assumption of a total surface free energy gamma is distributed components gamma d, and consists of the sum of the dipole component gamma p.
  • components of the gamma l are used two kinds of known liquid (pure water and diiodomethane in the present invention), measuring the respective contact angle ⁇ with respect to the scaffold for stem cell culture, gamma s d, simultaneous equations for gamma s p
  • the dispersion component ⁇ d and the dipole component ⁇ p of the scaffold for stem cell culture can be obtained by solving
  • the contact angle of the pure water is obtained by depositing 1 ⁇ L of pure water on the scaffold material and photographing the droplet image after 30 seconds using a contact angle meter (DMo-701, manufactured by Kyowa Interface Chemical Co., Ltd.) be able to.
  • the contact angle of diiodomethane can be obtained by depositing 1 ⁇ L of diiodomethane on the scaffold material and similarly photographing a droplet image after 30 seconds.
  • the scaffold material for stem cell culture preferably contains a synthetic resin from the viewpoint that the dispersion component ⁇ d of the surface free energy and the dipole component ⁇ p can be suitably adjusted.
  • the synthetic resin contains at least one of a polyvinyl acetal skeleton and a poly (meth) acrylic acid ester skeleton from the viewpoint that the dispersion component ⁇ d of the surface free energy and the dipole component ⁇ p can be suitably adjusted.
  • FIG. 1 is a diagram summarizing the relationship of the dipolar component ⁇ p to the dispersion component ⁇ d of the surface free energy of the main synthetic resin.
  • 2 and 3 are each a partially enlarged view of FIG.
  • the dispersion component ⁇ d of the surface free energy of the scaffold material for stem cell culture of the present invention is 24.5 or more and less than 45.0.
  • the dispersion ingredients gamma d is more preferably 28.0 or more 38.0 or less, more preferably 32.8 or more 36.0 or less.
  • the dipole component ⁇ p of the surface free energy of the scaffold material for stem cell culture of the present invention is 1.0 or more and less than 20.0.
  • As for the said dipole component (gamma) p 1.0 or more and 10.0 or less are more preferable, and 2.5 or more and 5.0 or less are more preferable.
  • the dispersion ingredients gamma d and the dipole component gamma p is, for example, can be controlled by changing the backbone of the synthetic resin described below as appropriate.
  • the dispersion component ⁇ d can be increased, for example, by increasing the amount of nonpolar functional groups or introducing a functional group having a cyclic structure from the skeleton of the synthetic resin, and a butyl group component in the synthetic resin Can be reduced by reducing the amount of
  • the synthetic resin preferably contains at least one of a polyvinyl acetal skeleton and a poly (meth) acrylic acid ester skeleton.
  • the dipole component ⁇ p can increase, for example, the amount of polar functional groups or the functional group including an ether structure from the skeleton of a synthetic resin and increase the amount of butyl groups which are nonpolar functional groups. Can be made smaller.
  • the synthetic resin refers to a resin having as a main component a polymer (hereinafter, also simply referred to as a “polymer”) obtained by polymerizing (also referred to as simply “a monomer”) of a polymerizable monomer (hereinafter also referred to as “monomer”).
  • the above polymers also include copolymers of one or more polymerizable monomers.
  • polymer examples include (unsaturated) hydrocarbons, aromatic hydrocarbons, (unsaturated) fatty acids, aromatic carboxylic acids, (unsaturated) ketones, aromatic ketones, (unsaturated) alcohols, aromatic alcohols, Examples thereof include polymers composed of one or more polymerizable monomers such as (un) saturated amines, aromatic amines, (unsaturated) thiols, aromatic thiols, and organosilicon compounds.
  • polystyrene resin examples include polyolefin, polyether, polyvinyl alcohol, polyvinyl acetal, polyester, poly (meth) acrylic acid ester, epoxy resin, polyamide, polyimide, polyurethane, polycarbonate, cellulose, polypeptide and the like.
  • poly (meth) acrylic acid esters and polyvinyl acetals are preferable, and polyvinyl acetals are more preferable, from the viewpoint of fixation of stem cells.
  • These polymers may be used alone or in combination of two or more.
  • two or more types of polymers When two or more types of polymers are combined, two or more types of polymers may be mixed and used, or may be used as a polymer in which the backbones of two or more types of polymers are chemically bonded.
  • two or more types of polymers When two or more types of polymers are combined as a synthetic resin, it is preferable to combine poly (meth) acrylic acid ester and polyvinyl acetal.
  • (meth) acrylic acids refers to at least one selected from the group consisting of (meth) acrylic esters and (meth) acrylic acids.
  • Poly (meth) acrylic acids are polymers obtained by polymerizing (meth) acrylic acid ester or (meth) acrylic acid which is the monomer, but (meth) acrylic acid ester or (meth) acrylic acid The thing which copolymerized other monomers is also included.
  • the (meth) acrylic acid ester is not particularly limited, but (meth) acrylic acid alkyl ester, (meth) acrylic acid cyclic alkyl ester, (meth) acrylic acid aryl ester, (meth) acrylamides, (meth) acrylic acid Polyethylene glycols, phosphoryl choline (meth) acrylate and the like can be mentioned.
  • Examples of the (meth) acrylic acid alkyl ester include methyl (meth) acrylate, ethyl (meth) acrylate, n-propyl (meth) acrylate, isopropyl (meth) acrylate, n-butyl (meth) acrylate, isobutyl (meth) ) Acrylates, t-Butyl (meth) acrylates, n-octyl (meth) acrylates, isooctyl (meth) acrylates, 2-ethylhexyl (meth) acrylates, nonyl (meth) acrylates, isononyl (meth) acrylates, decyl (meth) acrylates And isodecyl (meth) acrylate, lauryl (meth) acrylate, stearyl (meth) acrylate, isotetradecyl (meth) acrylate and the like.
  • the (meth) acrylic acid alkyl ester is not particularly limited, but may be substituted by various substituents such as an alkoxy group having 1 to 3 carbon atoms and a tetrahydrofurfuryl group. Examples include methoxyethyl acrylate, tetrahydrofurfuryl acrylate and the like.
  • Examples of the (meth) acrylic acid cyclic alkyl ester include cyclohexyl (meth) acrylate and isobornyl (meth) acrylate.
  • Examples of the (meth) acrylic acid aryl ester include phenyl (meth) acrylate, benzyl (meth) acrylate and the like.
  • acrylamides examples include (meth) acrylamide, N-isopropyl (meth) acrylamide, N-tert-butyl (meth) acrylamide, N, N'-dimethyl (meth) acrylamide, (3- (meth) acrylamide propyl ) Trimethylammonium chloride, 4- (meth) acryloyl morpholine, 3- (meth) acryloyl-2-oxazolidinone, N- [3- (dimethylamino) propyl] (meth) acrylamide, N- (2-hydroxyethyl) (meth) And the like) acrylamide, N-methylol (meth) acrylamide, 6- (meth) acrylamide hexanoic acid and the like.
  • Examples of the (meth) acrylic acid polyethylene glycols include methoxy-polyethylene glycol (meth) acrylate, ethoxy-polyethylene glycol (meth) acrylate, hydroxy-polyethylene glycol (meth) acrylate, methoxy-diethylene glycol (meth) acrylate, and ethoxy -Diethylene glycol (meth) acrylate, hydroxy-diethylene glycol (meth) acrylate, methoxy-triethylene glycol (meth) acrylate, ethoxy-triethylene glycol (meth) acrylate, hydroxy-triethylene glycol (meth) acrylate etc.
  • (meth) acrylic acid phosphoryl choline examples include 2- (meth) acryloyl oxyethyl phosphoryl choline and the like.
  • monomers other than (meth) acrylic acid ester (meth) acrylic acid, ethylene, vinyl ester etc. are mentioned.
  • the (meth) acrylic acid esters may be used alone or in combination of two or more.
  • the above (meth) acrylic acid is a generic term of acrylic acid and methacrylic acid
  • the above (meth) acrylate is a generic term of acrylate and methacrylate.
  • the first aspect of the present invention is preferably a combination of the second aspects described below from the viewpoint of enhancing the fixability of stem cells.
  • the second aspect of the present invention is a scaffold material for stem cell culture containing a synthetic resin, wherein the synthetic resin comprises a polyvinyl acetal resin, and the scaffold for stem cell culture has a degree of acetalization of polyvinyl acetal resin higher than 60 mol%. It relates to the material.
  • the scaffold material for stem cell culture of the present invention includes an embodiment comprising only a synthetic resin.
  • the scaffold material for stem cell culture has appropriate hydrophilicity and strength, the fixation after seeding of stem cells is improved.
  • the initial retention rate after stem cell seeding is improved.
  • the polyvinyl acetal resin is a resin synthesized by acetalizing polyvinyl alcohol with an aldehyde, and has an acetyl group, a hydroxyl group and an acetal group in the side chain.
  • the lower limit of the degree of acetalization of the polyvinyl acetal resin is preferably 60 mol%, and more preferably 90 mol%.
  • the degree of acetalization is 60% by mole or more, stem cell fixability is excellent and cell proliferation can be performed with high efficiency.
  • the solubility to a solvent can be made favorable as the degree of acetalization is 90 mol% or less.
  • a more preferable lower limit is 65 mol%, and a more preferable upper limit is 85 mol%.
  • the degree of acetal of the polyvinyl acetal resin can be measured by 1 H-NMR measurement.
  • aldehyde used for the acetalization examples include aldehydes having a linear aliphatic group having 1 to 10 carbon atoms, a cyclic aliphatic group or an aromatic group. As these aldehydes, conventionally known aldehydes can be used.
  • aldehyde is not particularly limited, but formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, pentanal, hexanal, heptanal, octanal, nonanal, decanal, acrolein, benzaldehyde, cinnamaldehyde, perylaldehyde, formyl pyridine, formyl imidazole, formyl pyrrole Formylpiperidine, formylpiperidine, formyltriazole, formyltetrazole, formylindole, formyl isoindole, formylpurine, formylpurine, formylbenzimidazole, formylbenzotriazole, formylquinoline, formyl isoquinoline, formylquinoxaline, formylcinnoline, formylpteridine, Formylfuran, Holmy Oxolane,
  • the aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde or pentanal, and more preferably butyraldehyde.
  • the lower limit of the polymerization degree of the polyvinyl acetal resin is preferably 100, more preferably 200, still more preferably 500, and still more preferably 1500.
  • the upper limit of the polymerization degree is preferably 6000, more preferably 3000, and still more preferably 2500.
  • the polyvinyl alcohol may be a copolymer with a vinyl compound.
  • the vinyl compound include ethylene, allylamine, vinylpyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth) acrylic acid, vinylamine, (meth) acrylic acid ester and the like.
  • said (meth) acrylic acid ester the (meth) acrylic acid ester mentioned above can be used, for example.
  • the polyvinyl acetal resin may be a graft copolymer with a vinyl compound.
  • a vinyl compound the above-mentioned compound is mentioned.
  • the graft copolymer contains a graft copolymer (hereinafter, also simply referred to as "graft copolymer”) having "a unit composed of a polyvinyl acetal" and a "unit composed of a vinyl compound".
  • the "unit made of polyvinyl acetal” and the "unit made of vinyl compound” refer to the "polyvinyl acetal" and the "unit made of vinyl compound” present in the graft copolymer.
  • a graft copolymer having a unit composed of a unit composed of polyvinyl acetal and a unit composed of a unit composed of a vinyl compound is a “unit composed of polyvinyl acetal” constituting a main chain or a “unit composed of a vinyl compound”
  • branched copolymer refers to a “polyvinyl acetal unit” or “vinyl compound unit” constituting different side chains.
  • the molecular weight of the graft copolymer is not particularly limited, but the number average molecular weight (Mn) is 10000 to 600000, the weight average molecular weight (Mw) is 20000 to 1200000, and the ratio (Mw / Mn) thereof is 2.%. It is preferably from 0 to 40. When the Mn, Mw, and Mw / Mn are in such ranges, the strength of the stem cell scaffold material is suitably maintained.
  • the degree of acetalization in the above graft copolymer can be obtained, for example, by dissolving the soluble matter in xylene of the above graft copolymer in deuterated dimethyl sulfoxide and measuring the degree of acetalization by 1 H-NMR measurement.
  • the polyvinyl acetal resin preferably has a Bronsted basic group or a Bronsted acidic group in part. That is, it is preferable that a part of the polyvinyl acetal resin is modified with a Bronsted basic group or a Bronsted acidic group, and it is more preferable that a part of the polyvinyl acetal resin is modified with a Bronsted basic group.
  • a part of the polyvinyl acetal resin is denatured by a Br ⁇ ⁇ ⁇ ⁇ nsted basic group or Br ⁇ ⁇ ⁇ nsted acid group, the initial retention rate after seeding with stem cells is improved in a serum-free medium culture containing no feeder cells or adhesion proteins, and stem cells Cultivation of
  • a polyvinyl acetal resin having a Bronsted basic group or a Bronsted acidic group in part of the polyvinyl acetal resin is referred to as a modified polyvinyl acetal resin.
  • the Br ⁇ ⁇ ⁇ ⁇ ⁇ nsted basic group is a generic term for functional groups that can receive hydrogen ion H + from other substances.
  • Examples of the Br ⁇ ⁇ ⁇ ⁇ ⁇ nsted basic group include amine basic groups such as a substituent having an amine structure, a substituent having an imine structure, a substituent having an amide structure, and a substituent having an imide structure. Therefore, as such a polyvinyl acetal resin, at least one selected from the group consisting of a structural unit having an amine structure, a structural unit having an imine structure, a structural unit having an amide structure, and a structural unit having an imide structure The polyvinyl acetal resin contained as a unit is preferable.
  • the total content of the structural unit having an amine structure, the structural unit having an imine structure, the structural unit having an amide structure, and the structural unit having an imide structure is 0.1 mol% to 30 mol% in the polyvinyl acetal resin It is preferably from 1 mol% to 10 mol% from the viewpoint of cell adhesion immediately after seeding.
  • the imine structure may be directly bonded to carbon constituting the main chain of the polyvinyl acetal resin, or may be bonded via a linking group such as an alkylene group.
  • having an imine structure in a side chain includes having the imine structure in a graft chain of a polyvinyl acetal resin.
  • the structural unit shown to following formula (1) is mentioned, for example.
  • R 1 represents a single bond or an alkylene group
  • R 2 represents a group having an imine structure
  • R 1 when R 1 is an alkylene group, the preferable lower limit of the carbon number of the alkylene group is 1, and the preferable upper limit is 12.
  • the carbon number of the above-mentioned alkylene group exceeds 12, optimum strength may not be obtained.
  • R ⁇ 1 > is an alkylene group, the more preferable upper limit of carbon number of the said alkylene group is five.
  • R 1 when R 1 is an alkylene group, examples of the alkylene group include methylene group, ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, octamethylene group, decamethylene group.
  • Linear alkylene groups such as methylmethylene group, methylethylene group, 1-methylpentylene group, branched alkylene group such as 1,4-dimethylbutylene group, cyclopropylene group, cyclobutylene group, cyclohexylene group, etc.
  • cyclic alkylene groups of the following Among them, linear alkyl groups such as methylene, ethylene, trimethylene and tetramethylene are preferable, and methylene and ethylene are more preferable.
  • R 2 it includes a functional group represented by the following formula (2).
  • R 3 represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms
  • R 4 represents a hydrocarbon group having 1 to 18 carbon atoms.
  • hydrocarbon group a saturated hydrocarbon group, an unsaturated hydrocarbon group, an aromatic hydrocarbon group etc. are mentioned.
  • the above-mentioned hydrocarbon group may consist of only one kind of a saturated hydrocarbon group, an unsaturated hydrocarbon group, and an aromatic hydrocarbon group, and two or more kinds of these are used. May be
  • saturated hydrocarbon group examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, iso-butyl group, sec-butyl group, tert-butyl group, pentyl group and hexyl group. And heptyl group, 2-ethylhexyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, octadecyl group and the like.
  • methyl group, ethyl group, n-propyl group and n-butyl group are preferable.
  • the aromatic hydrocarbon group include phenyl group, toluyl group, xylyl group, t-butylphenyl group, benzyl group and the like.
  • R 1 is a single bond
  • R 3 is a hydrogen atom, a methyl group or an ethyl group
  • R 4 is a methyl group, an ethyl group or a propyl group in structural units having the imine structure.
  • the preferable lower limit of content of the structural unit which has an imine structure is 0.1 mol%, and a preferable upper limit is 20.0 mol%.
  • a preferable upper limit is 20.0 mol%.
  • the content of the structural unit having an imine structure is 0.1 mol% or more, the viscosity stability over time becomes good.
  • Acetalization can fully be advanced as content of the structural unit which has the said imine structure is 20.0 mol% or less.
  • the more preferable lower limit of the content of the constituent unit having an imine structure is 1.0 mol%, and the more preferable upper limit is 15.0 mol%.
  • the content of the constituent unit having the imine structure can be measured by 1 H-NMR measurement.
  • the ratio of the content of the structural unit having an imine structure to the degree of acetalization described later is preferably 0.001 to 0.5. .
  • the imino group may be directly bonded to carbon constituting the main chain of the polyvinyl acetal resin, or may be bonded via a linking group such as an alkylene group.
  • the modified polyvinyl acetal resin preferably has a structural unit having an amine structure or a structural unit having an amide structure. It is preferable that the said modified polyvinyl acetal resin has the said amine structure or an amide structure in a side chain.
  • the above amine structure or amide structure may be directly bonded to carbon constituting the main chain of the modified polyvinyl acetal resin, or may be bonded via a linking group such as an alkylene group.
  • the above amine structure may be a primary amine, a secondary amine, a tertiary amine or a quaternary amine. Among these, primary amines are preferred from the viewpoint of enhancing the fixability of stem cells.
  • having the above-mentioned amine structure or an amide structure in a side chain means having the above-mentioned amine structure or an amide structure in the graft chain of denaturation polyvinyl acetal resin.
  • the amine structure is preferably -NH 2 .
  • the structural unit which has the said amine structure is a structure shown to following formula (3).
  • the structural unit which has the said amide structure is a structure shown to following formula (4).
  • R 5 represents a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms.
  • hydrocarbon group an alkyl group, an alkenyl group, a cycloalkyl group, and a cycloalkenyl group are mentioned.
  • the preferable lower limit of the content of the constituent unit having an amine structure or an amide structure is 0.1 mol%, and the preferable upper limit is 20 mol%.
  • the content of the constituent unit having the amine structure or the amide structure is 0.1 mol% or more, the addition property can be made sufficient.
  • the content is 20 mol% or less, the solubility does not increase excessively, and the removal of the modified polyvinyl acetal resin powder by the precipitation method becomes easy.
  • the more preferable lower limit of the content is 0.5 mol%, and the more preferable upper limit is 10 mol%.
  • the content of the structural unit having the above amine structure or amide structure can be measured by 1 H-NMR measurement.
  • the preferable lower limit of the content which totaled the structural unit which has the said amine structure or an amide structure, and the structural unit which has an imine structure is 0.1 mol%, and a preferable upper limit is 20 mol%.
  • the more preferable lower limit of the content is 0.5 mol%, and the more preferable upper limit is 10 mol%.
  • the content ratio of the structural unit having an imine structure to the structural unit having an amine structure or an amide structure is 0 It is preferably 5 / 99.5 to 99.5 / 0.5.
  • the above ratio is 0.5 / 99.5 or more, the viscosity stability over time can be made sufficient, and when the above ratio is 99.5 / 0.5 or less, the stem cell fixability can be improved.
  • the crosslinking performance can be sufficiently exhibited from the viewpoint.
  • the more preferable lower limit of the above ratio is 5/95, and the more preferable upper limit is 90/10.
  • the Br ⁇ ⁇ ⁇ ⁇ nsted acid group is a generic term for functional groups that can transfer hydrogen ion H + to other substances.
  • the Bronsted acidic group include a carboxyl group, a sulfonic acid group, a maleic acid group, a sulfinic acid group, a sulfenic acid group, a phosphoric acid group, a phosphonic acid group, and salts thereof.
  • a carboxyl group is preferable as the Bronsted acidic group.
  • the method for modifying the polyvinyl acetal resin with the Br ⁇ ⁇ ⁇ ⁇ nsted acidic group is not particularly limited, but a method of copolymerizing the polyvinyl alcohol with the itaconic acid or (meth) acrylic acid, Br ⁇ ⁇ ⁇ ⁇ nsted acid on the side chain of the polyvinyl alcohol It is obtained by a method of introducing a group, etc.
  • the degree of acetalization of the polyvinyl acetal resin is not particularly limited, but the preferable lower limit is 60 mol%, and the preferable upper limit is 90 mol%. When the degree of acetalization is 60% by mole or more, stem cell fixability is excellent and cell proliferation can be performed with high efficiency. Moreover, the solubility to a solvent can be made favorable as the degree of acetalization is 90 mol% or less. A more preferable lower limit is 65 mol%, and a more preferable upper limit is 85 mol%.
  • the degree of acetal of the polyvinyl acetal resin can be measured by 1 H-NMR measurement.
  • the amount of acetyl groups in the polyvinyl acetal resin is not particularly limited, and the preferable lower limit is 0.0001% by mol, and the preferable upper limit is 5% by mol.
  • the polyvinyl alcohol obtained by saponifying the polyvinyl acetate obtained by copolymerizing the monomer which has the said imine structure, and vinyl acetate is known conventionally, for example
  • the method of acetalization by the method of is mentioned.
  • a method of introducing an imine structure by acetalizing a polyvinyl alcohol having a structural unit having an amino group or an amide structure by a conventionally known method may be used.
  • the modified polyvinyl alcohol having an imine structure obtained by post-modifying a polyvinyl alcohol having a structural unit having an amino group or an amide structure may be acetalized by a conventionally known method.
  • the imine structure may be introduced by post-denaturing the unmodified polyvinyl acetal resin.
  • the modified polyvinyl acetal resin may be an acetalized polyvinyl alcohol having a structural unit having an amino group or an amide structure.
  • a method of obtaining a modified polyvinyl acetal resin having an imine structure by acetalizing a polyvinyl alcohol having a structural unit having an amino group or an amide structure is preferable.
  • an imine structure can be obtained by adding an excess amount of an aldehyde and an acid catalyst used for acetalization.
  • aldehyde in excess, it is preferable to add 70 to 150 parts by weight of aldehyde to 100 parts by weight of polyvinyl alcohol having a structural unit having an amino group or an amide structure.
  • aldehyde acetaldehyde, propionaldehyde, n-butyraldehyde, isobutyraldehyde, n-valeraldehyde and phenylaldehyde are preferable.
  • the acid catalyst In the method of adding the acid catalyst in excess, it is preferable to add the acid catalyst by 0.5% by weight or more of the whole. Furthermore, it is preferable to add 5.0 to 70.0 parts by weight of an acid catalyst to 100 parts by weight of polyvinyl alcohol having a structural unit having an amino group or an amide structure. In particular, hydrochloric acid, nitric acid, sulfuric acid and paratoluenesulfonic acid are preferable as the acid catalyst.
  • examples of a method of confirming a structural unit having an amino group, an amide structure, and a structural unit having an imine structure include a method of confirming by 1 H-NMR.
  • the acetalization can be carried out using a known method, and is preferably carried out in an aqueous solvent, a mixed solvent of water and an organic solvent compatible with water, or an organic solvent.
  • an organic solvent compatible with water for example, an alcohol-based organic solvent can be used.
  • the organic solvent include alcohol organic solvents, aromatic organic solvents, aliphatic ester solvents, ketone solvents, lower paraffin solvents, ether solvents, amine solvents and the like.
  • the alcohol-based organic solvent include methanol, ethanol, n-propanol, isopropanol, n-butanol, tert-butanol and the like.
  • Examples of the aromatic organic solvent include xylene, toluene, ethylbenzene, methyl benzoate and the like.
  • Examples of the aliphatic ester solvents include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, ethyl propionate, methyl butyrate, ethyl butyrate, methyl acetoacetate, ethyl acetoacetate and the like.
  • Examples of the ketone solvents include acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, methyl cyclohexanone, benzophenone, acetophenone and the like.
  • Examples of the lower paraffinic solvents include hexane, pentane, octane, cyclohexane and decane.
  • Examples of the ether solvents include diethyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol diethyl ether and the like.
  • Examples of the amide solvents include N, N-dimethylformamide, N, N-dimethyl tecetamide, N-methyl pyrrolidone, acetanilide and the like.
  • amine solvents examples include ammonia, trimethylamine, triethylamine, n-butylamine, di-n-butylamine, tri-n-butylamine, aniline, N-methylaniline, N, N-dimethylaniline and pyridine. These can be used alone or in combination of two or more solvents. Among these, ethanol, n-propanol, isopropanol and tetrahydrofuran are particularly preferable from the viewpoint of solubility in a resin and ease of purification.
  • the acetalization is preferably performed in the presence of an acid catalyst.
  • the above-mentioned acid catalyst is not particularly limited, and mineral acids such as sulfuric acid, hydrochloric acid, nitric acid and phosphoric acid, carboxylic acids such as formic acid, acetic acid and propionic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid and paratoluene sulfone And sulfonic acids such as acids.
  • These acid catalysts may be used alone or in combination of two or more. Among them, hydrochloric acid, nitric acid and sulfuric acid are preferable, and hydrochloric acid is particularly preferable.
  • the above-mentioned stem cell type is not particularly limited by using the stem cell scaffold material of the present invention, but it can be used as a stem cell scaffold material. Among them, it is preferable to be used for culturing pluripotent stem cells, particularly iPS cells. In a serum-free medium culture that does not contain feeder cells or adhesion proteins, the initial retention rate after seeding with stem cells is improved, and stem cells can be cultured preferably.
  • pluripotent stem cells are considered to have a low establishment rate in culture after seeding, but the above scaffold material for stem cell culture is not easily swelled by the water of the culture medium, and maintains adequate hydrophilicity and strength This is because the establishment rate after seeding of pluripotent stem cells is improved.
  • the scaffold material for stem cell culture is, in addition to use for planar culture (two-dimensional culture method) in stem cell culture, a state closer to in vivo, for example, culture of stem cells on a substrate such as porous membrane or hydrogel It can be used for (three-dimensional culture method). This is because stem cells can be efficiently propagated by using a scaffold for cell culture for a bioreactor or the like.
  • the scaffold material for cell culture is preferably used in a two-dimensional culture method because it has appropriate hydrophilicity and strength.
  • the shape and size of the container for planar culture are not particularly limited, and examples thereof include a cell culture test plate provided with one or more wells and a cell culture flask. Although the number of wells of the above-mentioned microplate is not limited, for example, 2, 4, 6, 12, 24, 48, 96, 384 and the like can be mentioned.
  • the shape of the well is not particularly limited, and examples thereof include a perfect circle, an ellipse, a triangle, a square, a rectangle, a pentagon, and the like.
  • the shape of the bottom of the well is not particularly limited, and examples thereof include a flat bottom, a round bottom, and irregularities.
  • the material of the cell culture test plate provided with one or more wells and the cell culture flask is not particularly limited, and examples thereof include polymer resins, metals, and inorganic materials.
  • the polymer resin include polystyrene, polyethylene, polypropylene, polycarbonate, polyester, polyisoprene, cycloolefin polymer, polyimide, polyamide, polyamide imide, (meth) acrylic resin, epoxy resin, silicone and the like.
  • the metal include stainless steel, copper, iron, nickel, aluminum, titanium, gold, silver, platinum and the like.
  • the inorganic material include silicon oxide (glass), aluminum oxide, titanium oxide, zirconium oxide, iron oxide, silicon nitride and the like.
  • the scaffold for cell culture can be used in a suspension culture method in which stem cells are freely suspended and grown in a culture medium.
  • Method of culturing pluripotent stem cells In the method for culturing pluripotent stem cells, it is preferable to seed the cell mass on a scaffold for stem cell culture containing a synthetic resin.
  • a cell mass can be obtained by adding a cell release agent to a culture vessel that has become confluent and disrupting the cells uniformly by pipetting.
  • the cell release agent is not particularly limited, but is preferably an ethylenediamine / phosphate buffer solution.
  • the size of the cell mass is preferably 50 to 200 ⁇ m.
  • the present invention provides an invention using a scaffold material for stem cell culture as another embodiment in addition to the scaffold material for stem cell culture described above.
  • a stem cell culture carrier (medium) containing the above-described stem cell culture scaffold material and a polysaccharide.
  • polysaccharides various polysaccharides can be used without particular limitation. Among them, water-soluble polysaccharides are preferred.
  • a container for stem cell culture comprising a resin film in at least a part of a cell culture region, wherein the stem cell culture container using the scaffold material for stem cell culture described above as the resin film is provided.
  • the container is not particularly limited as long as it has a resin film on at least a part of the cell culture area, and various containers can be used.
  • the container for flat culture mentioned above, a bioreactor, etc. can be used.
  • a fiber for stem cell culture comprising a scaffold material for stem cell culture.
  • the scaffold material for stem cell culture is preferably coated on a fiber.
  • the scaffold material for stem cell culture may be in the form of being impregnated or kneaded into fibers.
  • the stem cell culture fiber is difficult to adhere to a planar structure such as a flask, but is suitable for a three-dimensional culture method of stem cells that easily adheres to a three-dimensional structure such as a fibril-like structure.
  • stem cells it is particularly suitable for culturing adipose stem cells.
  • the scaffold material for stem cell culture may be crosslinked. It is because water swelling property can be suppressed and strength can be raised suitably by being bridged.
  • a crosslinking agent may be further added to the scaffold material for stem cell culture to cause crosslinking.
  • the crosslinking agent is not particularly limited, and examples thereof include polyalcohols, polycarboxylic acids, hydroxycarboxylic acids, metal soaps, polysaccharides and the like.
  • the polyalcohol is not particularly limited, but ethylene glycol, propylene glycol, butanediol, pentanediol, hexanediol, heptanediol, octanediol, nonanediol, decanediol, dodecanediol, undecanediol, undecanediol, diethylene glycol, triethylene glycol, tetraethylene
  • examples thereof include glycol, polyethylene glycol, catechol, pyrogallol, diboronic acid, methylene diboronic acid, ethylene diboronic acid, propylene diboronic acid, phenylene diboronic acid, biphenyl diboronic acid, and bisphenol derivatives.
  • the polycarboxylic acid is not particularly limited, and oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, phthalic acid, poly (meth) acrylic acid and the like can be mentioned. .
  • the hydroxycarboxylic acid is not particularly limited, but glycolic acid, lactic acid, thalthronic acid, glyceric acid, hydroxybutyric acid, malic acid, tartaric acid, tartaric acid, citric acid, isocitric acid, leucine acid, mevalonic acid, pantoic acid, ricinoleic acid, Ricinereidiic acid, cerebronic acid, quinic acid, shikimic acid, hydroxybenzoic acid, salicylic acid, creosote acid, vanillic acid, silicic acid, pyrocatechic acid, resorcylic acid, protocatechuic acid, gentisic acid, orsellinic acid, gallic acid, mandelic acid, benzyl Acids, atrolactic acid, melilotonic acid, phlorettic acid, coumaric acid, umbellic acid, caffeic acid, ferulic acid, sinapinic acid, hydroxystearic acid and the like can be mentioned.
  • the metal soap is not particularly limited, and examples thereof include fatty acids such as stearic acid, lauric acid, ricinoleic acid and octylic acid, and salts of metals such as lithium, sodium, magnesium, calcium, barium, zinc and aluminum.
  • the polysaccharides are not particularly limited, but pectin, guar gum, xanthan gum, tamarind gum, carrageenan, propylene glycol, carboxymethylcellulose, amylose, amylopectin, glycogen, cellulose, chitin, agarose, carrageenan, heparin, hyaluronic acid, xyloglucan, glucomannan An acid etc. are mentioned.
  • the content of the structural unit in the obtained synthetic resin and modified polyvinyl acetal resin for example, the structural unit having an amine structure (mol%), the content of the structural unit having an imine structure (mol%), and an amide structure
  • the content (mol%) of the constituent units, the degree of acetalization (mol%), the amount of acetyl groups (mol%), the amount of hydroxyl groups (mol%), and the amount of (meth) acrylic acid ester groups (mol%) It was dissolved in DMSO-d6 (dimethyl sulfoxide) and measured using 1 H-NMR (nuclear magnetic resonance spectrum).
  • Example 1 (Preparation of polyvinyl butyral)
  • 300 g of polyvinyl alcohol having 2700 mL of ion-exchanged water, an average polymerization degree of 250, and a saponification degree of 99 mol% was charged and dissolved with heating while stirring to obtain a solution.
  • 35 wt% hydrochloric acid as a catalyst is added to this solution so that the concentration of hydrochloric acid is 0.2 wt%, the temperature is adjusted to 15 ° C., and n-butyraldehyde (n-BA) is stirred. 22 g was added.
  • n-butyraldehyde (n-BA) was added to precipitate white particles of polyvinyl butyral. 15 minutes after precipitation, 35 wt% hydrochloric acid was added so that the concentration of hydrochloric acid was 1.8 wt%, heated to 50 ° C., and aged at 50 ° C. for 2 hours. Then, the solution was cooled and neutralized, and then polyvinyl butyral was washed with water and dried to obtain polyvinyl butyral. The obtained polyvinyl butyral had an average degree of polymerization of 250, an amount of hydroxyl groups of 28 mol%, an amount of acetyl groups of 1 mol%, and an acetalization degree of 71 mol%.
  • a polyvinyl butyral solution was obtained by dissolving 1 g of the obtained polyvinyl butyral in 19 g of butanol. Discharge 150 ⁇ L of the obtained polyvinyl butyral solution onto a ⁇ 22 mm cover glass (Matsunami Co., Ltd., using No. 1 dust removed with an air duster) and spin it at 2000 rpm for 20 seconds using a spin coater to make it smooth A resin film was obtained. The obtained resin film was placed on a polystyrene dish with a diameter of 22 mm together with the cover glass to obtain a cell culture vessel.
  • the surface free energy of the resin film was measured using a contact angle meter (DMo-701, manufactured by Kyowa Interface Chemical Co., Ltd.). A contact angle of pure water was obtained by depositing 1 ⁇ l of pure water on the resin film and photographing a droplet image after 30 seconds. Further, 1 ⁇ L of diiodomethane was deposited on the resin film, and a droplet image after 30 seconds was taken to obtain a contact angle of diiodomethane.
  • the surface free energy ⁇ , the dispersion component ⁇ d , and the dipole component ⁇ p were derived from the obtained contact angles using the Kaelble-Uy theory.
  • Tests were conducted on a cell culture vessel provided with a resin film under the following conditions.
  • 1 mL of phosphate buffered saline was added to the obtained cell culture vessel, and the cells were allowed to stand in a 37 ° C. incubator for 1 hour.
  • 1.5 ⁇ 10 4 h-iPS cells 253G1 are seeded, 1 mL of medium TeSR E8 (STEM CELL) and 10 ⁇ M of ROCK-Inhibitor (Y27632) are present.
  • Culturing was carried out at 37 ° C. in a 5% CO 2 incubator. The medium was changed by removing 750 ⁇ l of the medium every 24 hours, adding 250 ⁇ l of fresh TeSR E8, and adjusting to 10 ⁇ M of ROCK-Inhibitor (Y27632).
  • TeSRE 8 medium is inoculated with 1.0 ⁇ 10 5 cells into a culture vessel, and medium TeSR E8 (STEM CELL Co., Ltd.) 2.
  • Cultivation was carried out in the presence of 1 mL and 10 ⁇ M of ROCK-Inhibitor (Y27632) at 37 ° C. in a 5% CO 2 incubator. The medium was changed by removing 750 ⁇ l of the medium every 24 hours and adding 250 ⁇ l of fresh TeSR E8.
  • Adhesion maintenance property In the cell mass culture test, adhesion maintenance possible time of cell mass was evaluated according to the following criteria. 0: All cells detached in less than 30 minutes after medium replacement. 1: Adhesion was maintained for 30 minutes or more after medium replacement, but all cells were detached in less than 1 hour. 2: Adhesion was maintained for 1 hour or more after medium replacement, but all cells were detached in less than 24 hours. 3: Adhesion was maintained for 24 hours or more after medium replacement. The obtained cell mass was confirmed to be undifferentiated by alkaline phosphatase (ALP) staining test.
  • ALP alkaline phosphatase
  • Example 2 The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average polymerization degree of 850 and a saponification degree of 99 mol% was used.
  • Example 3 The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average degree of polymerization of 1700 and a degree of saponification of 99 mol% was used.
  • Example 4 The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average degree of polymerization of 2400 and a degree of saponification of 99 mol% was used, and that acetaldehyde was used instead of n-butyraldehyde (n-BA).
  • n-BA n-butyraldehyde
  • Example 5 The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average polymerization degree of 850, a saponification degree of 98 mol% and an ethylene modification degree of 4 mol% was used.
  • Example 6 The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average degree of polymerization of 250, a degree of saponification of 99 mol%, and 2 mol% of a constituent unit having an amino group shown in the above formula (3) was used.
  • Example 7 The test was conducted in the same manner as in Example 1 except that a polyvinyl alcohol having an average polymerization degree of 1600, a saponification degree of 99 mol%, and a structural unit having an amino group shown in the above formula (3) was used.
  • Example 8 100 parts by weight of polyvinyl acetal having a degree of polymerization of about 250 obtained in Example 1 and 1 part by weight of N-vinylpyrrolidone were dissolved in 500 parts by weight of tetrahydrofuran to obtain a graft copolymer resin solution.
  • 0.05 parts by weight of Irgacure 184 manufactured by BASF Corporation was dissolved and applied onto a PET film.
  • a composite resin solution was obtained by irradiating a light having a wavelength of 365 nm with an integrated light quantity of 2000 mJ / cm 2 using a UV conveyor apparatus “ECS 301 G1” manufactured by Eye Graphics Co., Ltd. at 25 ° C.
  • the resulting composite resin solution was vacuum dried at 80 ° C. for 3 hours to obtain a composite resin. It was about 40,000 when the weight average molecular weight by polystyrene conversion was measured by GPC method using "2690 Separations Model" by Waters company about the obtained resin as a column. The obtained composite resin was adjusted to a 3% by weight butanol solution, and tested in the same manner as in Example 1.
  • Example 9 The test was conducted in the same manner as in Example 8 except that 10 parts by weight of N-vinylpyrrolidone was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 60,000.
  • Example 10 The test was conducted in the same manner as in Example 8 except that 30 parts by weight of N-vinylpyrrolidone was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 50,000.
  • Example 11 The test was conducted in the same manner as in Example 8 except that 5 parts by weight of tetrahydrofurfuryl acrylate was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 60,000.
  • Example 12 The test was conducted in the same manner as in Example 8 except that 5 parts by weight of methoxyethyl acrylate was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 70,000.
  • Example 13 The test was conducted in the same manner as in Example 8 except that 5 parts by weight of butyl methacrylate was added to 100 parts by weight of polyvinyl acetal.
  • the weight average molecular weight of the obtained resin is about 60,000.
  • Example 14 An acrylic monomer solution was obtained by dissolving 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate in 300 parts by weight of tetrahydrofuran. Two parts by weight of Irgacure 184 (manufactured by BASF Corp.) was dissolved in the obtained acrylic monomer solution, and the solution was coated on a PET film. An acrylic resin solution was obtained by irradiating a light having a wavelength of 365 nm with an integrated light quantity of 2000 mJ / cm 2 using a UV conveyor apparatus “ECS 301 G1” manufactured by Eye Graphics Co., Ltd. at 25 ° C. The obtained acrylic resin solution was vacuum dried at 80 ° C. for 3 hours to obtain an acrylic resin. The resulting acrylic resin was adjusted to a 3% by weight butanol solution, and tested in the same manner as in Example 1. The weight average molecular weight of the obtained acrylic resin was about 100,000.
  • Example 15 An acrylic resin was prepared in the same manner as in Example 14 except that 90 parts by weight of methoxyethyl acrylate and 10 parts by weight of butyl methacrylate were used instead of 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate. Obtained. The resulting acrylic resin was adjusted to a 3% by weight butanol solution, and tested in the same manner as in Example 1. The weight average molecular weight of the obtained acrylic resin was about 80,000.
  • Example 16 An acrylic resin was prepared in the same manner as in Example 14 except that 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate were used, and 75 parts by weight of methoxyethyl acrylate and 25 parts by weight of butyl methacrylate were used. Obtained. The resulting acrylic resin was adjusted to a 3% by weight butanol solution, and tested in the same manner as in Example 1. The weight average molecular weight of the obtained resin was about 90,000.
  • Example 17 An acrylic resin is obtained in the same manner as in Example 14 except that 2 parts by weight of butyl methacrylate and 98 parts by weight of ethyl acrylate are used instead of 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate. The The resulting acrylic resin was adjusted to a 3% by weight butanol solution, and tested in the same manner as in Example 1. The weight average molecular weight of the obtained acrylic resin was about 80,000.
  • Comparative Example 1 The test was performed in the same manner as in Example 1 using only a polystyrene dish without using a scaffold material.
  • Comparative Example 2 The test was performed in the same manner as in Example 1 except that the second addition amount of n-butyraldehyde (n-BA) was changed from 148 g to 89 g.
  • n-BA n-butyraldehyde
  • Comparative Example 3 The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average polymerization degree of 1000 and a saponification degree of 98 mol% was used as the synthetic resin.
  • Comparative Example 4 A polyacrylamide resin was obtained by mixing 100 parts by weight of N-isopropyl acrylamide, 75 parts by weight of ethyl acetate and 0.5 parts by weight of azobisisobutyronitrile, and performing polymerization at 65 ° C. for 8 hours in a nitrogen atmosphere. It was about 90,000 (degree of polymerization about 800) when the weight average molecular weight by polystyrene conversion was measured by GPC method using "2690 Separations Model" by Waters company about the obtained resin as a column. The other operations were tested in the same manner as in Example 1.
  • Comparative Example 5 The test was conducted in the same manner as Comparative Example 4 except that 100 parts by weight of ethyl acrylate was used instead of 100 parts by weight of N-isopropylacrylamide.
  • Comparative Example 6 The test was conducted in the same manner as Comparative Example 4 except that 100 parts by weight of butyl methacrylate was used instead of 100 parts by weight of N-isopropylacrylamide. The weight average molecular weight of the obtained resin was about 90,000.
  • Comparative Example 7 The test was conducted in the same manner as in Example 8 except that 70 parts by weight of N-vinylpyrrolidone was added to 30 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 90,000.
  • Tables 1 and 2 The obtained results are summarized in Tables 1 and 2.
  • the phase contrast micrographs of the cells after 24 hours after seeding are shown in FIG. 5 and FIG.
  • the phase contrast micrograph of the cell 5 days after seeding is shown in FIG. 8 and FIG.
  • differentiated cells were not observed in any of the examples and comparative examples.

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Abstract

A scaffolding material for stem cell cultures, having a dispersion component γd for surface free energy of at least 24.5 and less than 45.0 and a dipole component γp for surface free energy of at least 1 and less than 20.0. This scaffolding material for stem cell cultures provides suitable hydrophilic properties and strength, high fixation of stem cells after seeding, and highly efficient cell proliferation.

Description

幹細胞培養用足場材料及びそれを用いた幹細胞培養方法Scaffold material for stem cell culture and stem cell culture method using the same
　本発明は、幹細胞培養用足場材料及びそれを用いた幹細胞培養方法に関する。 The present invention relates to a scaffold material for stem cell culture and a stem cell culture method using the same.
　幹細胞は創薬や再生医療への応用が期待されている。幹細胞は、自己複製能と分化能を有する細胞であり、全ての細胞種へ分化可能な多能性幹細胞と、同系列の体組織の構成細胞種へのみ分化可能な組織幹細胞および組織前駆細胞がある。多能性幹細胞は、例えばヒト胚性幹細胞（ｈＥＳＣ）や、ヒト人工多能性幹細胞（ｈｉＰＳＣ）などのヒト多能性幹細胞（ｈＰＳＣ）等が挙げられる。幹細胞を安全に、そして再現性良く培養し、増殖させることは、これらの細胞を医療応用する上では必須の基盤技術となる。特に、再生医療の産業利用上においては、幹細胞を未分化状態で多量に扱う必要がある。そのため、天然および合成の高分子やフィーダー細胞を用いて幹細胞の増殖を行うと共に、多能性能（または多分化能）を維持する技術について広範な研究が行われている。特に天然高分子としてラミニン、ヴィトロネクチンなどの接着タンパク質やマウス肉腫由来のマトリゲルを使用すると播種後の細胞定着性が非常に高いことが知られている。 Stem cells are expected to be applied to drug discovery and regenerative medicine. Stem cells are cells having self-replication ability and differentiation ability, and pluripotent stem cells capable of differentiating into all cell types, and tissue stem cells and tissue precursor cells capable of differentiating only into constituent cell types of somatic body tissues. is there. Examples of pluripotent stem cells include human embryonic stem cells (hESCs) and human pluripotent stem cells (hPSCs) such as human induced pluripotent stem cells (hiPSCs). Culturing and expanding stem cells safely and reproducibly is an essential basic technology for medical application of these cells. In particular, in industrial use of regenerative medicine, it is necessary to treat stem cells in a large amount in an undifferentiated state. Therefore, extensive research has been conducted on techniques for maintaining stem cells with natural and synthetic macromolecules and feeder cells and maintaining pluripotency (or pluripotency). In particular, it is known that cell adhesion after seeding is extremely high when adhesion proteins such as laminin and vitronectin and matrigel derived from mouse sarcoma are used as natural polymers.
　しかし、天然高分子は生産性が非常に低いため高価であること、天然由来物質であるためロット間にバラツキが見られること、動物由来の成分による安全性上の懸念があることが課題として挙げられる。 However, natural polymers are very expensive because they have very low productivity, that they are naturally derived substances, there are lot-to-lot variations, and there are safety concerns due to animal-derived components. Be
　上記課題を解決するために、合成樹脂を使用した幹細胞培養樹脂担体が提案されている。例えば、特許文献１の実施例の欄には、マウス線維芽細胞の培養において、親水性かつ耐水性に優れる足場材を提供するためにアセタール化度が２０～６０モル％のポリビニルアセタール化合物が開示されている。特許文献２の実施例の欄にはマウスＥＳ細胞の培養において、アクリルポリマーによって構成されたハイドロゲルが開示されている。特許文献３の実施例の欄にはマウスｉＰＳ細胞の培養において、親水性かつ柔軟なポリロタキサンゲルが開示されている。 In order to solve the above-mentioned subject, a stem cell culture resin carrier using synthetic resin is proposed. For example, in the column of Example of Patent Document 1, disclosed is a polyvinyl acetal compound having a degree of acetalization of 20 to 60 mol% to provide a scaffold having excellent hydrophilicity and water resistance in culturing mouse fibroblasts. It is done. The column of Example of Patent Document 2 discloses a hydrogel composed of an acrylic polymer in the culture of mouse ES cells. The column of the example of Patent Document 3 discloses a hydrophilic and flexible polyrotaxane gel in the culture of mouse iPS cells.
特開２００６－３１４２８５号公報JP, 2006-314285, A 特開２０１０－１５８１８０号公報JP, 2010-158180, A 特開２０１７－２３００８号公報Unexamined-Japanese-Patent No. 2017-23008
　しかしながら、特許文献１には、親水性が高いため培地中で膨潤し、足場材樹脂が剥がれる問題がある。また、幹細胞や多能性幹細胞の播種後の定着性が低く、十分に増殖しないという問題がある。特許文献２には、２－アクリルアミド－２－メチルプロパンスルホン酸ナトリウム塩、および、ｐ－スチレンスルホン酸ナトリウム、Ｎ，Ｎ’－ジメチルアクリルアミドを使用しており、親水性が高いため培地中で膨潤し、足場材樹脂が剥がれる問題がある。特許文献３には、親水性が高いため培地中で膨潤し、足場材樹脂が剥がれる問題がある。また、柔軟な足場材であるために心筋細胞への分化が促進されるという問題がある。 However, Patent Document 1 has a problem that it swells in a culture medium due to its high hydrophilicity and the scaffold resin is peeled off. In addition, there is a problem that the fixation after seeding of stem cells and pluripotent stem cells is low, and the cells do not proliferate sufficiently. Patent Document 2 uses sodium salt of 2-acrylamido-2-methylpropane sulfonic acid and sodium p-styrene sulfonate, N, N'-dimethyl acrylamide, and has high hydrophilicity and thus swells in a medium. And there is a problem that the scaffold resin is peeled off. Patent Document 3 has a problem that it swells in a culture medium due to its high hydrophilicity and the scaffold resin is peeled off. In addition, there is a problem that the differentiation to cardiomyocytes is promoted because it is a flexible scaffold.
　以上より、適度な親水性と強度を備える幹細胞培養用足場材料及びそれを用いた幹細胞培養方法が求められていた。
　本発明は、適度な親水性と強度を備え、幹細胞の播種後の定着性が高く、高効率に細胞増殖が可能な幹細胞培養用足場材料及びそれを用いた幹細胞培養方法を提供することを目的とする。 From the above, a scaffold material for stem cell culture having appropriate hydrophilicity and strength and a stem cell culture method using the same have been desired.
An object of the present invention is to provide a scaffold material for stem cell culture which has appropriate hydrophilicity and strength, is highly stable after seeding of stem cells, and can efficiently proliferate cells, and a stem cell culture method using the same. I assume.
　本発明は以下の内容に関する。
（１）表面自由エネルギーの分散成分γｄが２４．５以上４５．０未満であり、表面自由エネルギーの双極子成分γｐが１．０以上２０．０未満である幹細胞培養用足場材料。
（２）幹細胞培養用足場材料が合成樹脂を含む、（１）記載の幹細胞培養用足場材料。
（３）合成樹脂がポリビニルアセタール骨格、ポリ（メタ）アクリル酸エステル骨格の少なくともいずれか一方を含む（２）記載の幹細胞培養用足場材料。
（４）合成樹脂がポリビニルアセタール樹脂である（２）記載の幹細胞培養用足場材料。
（５）合成樹脂を含有する幹細胞培養用足場材料であって、合成樹脂がポリビニルアセタール樹脂を含み、ポリビニルアセタール樹脂のアセタール化度が６０モル％よりも高い幹細胞培養用足場材料。
（６）ポリビニルアセタール樹脂は、イミン構造を有する構成単位、アミノ基を有する構成単位、及びアミド構造を有する構成単位からなる群より選択される少なくとも一種を有する、（４）または（５）の幹細胞培養用足場材料。
（７）ポリビニルアセタール樹脂は、イミン構造を有する構成単位、アミノ基を有する構成単位、及びアミド構造を有する構成単位を合計した含有量が、０．１モル％以上２０モル％以下である、（６）記載の幹細胞培養用足場材。
（８）幹細胞が多能性幹細胞である（１）～（７）のいずれか１項記載の幹細胞培養用足場材料。
（９）細胞の培養領域の少なくとも一部に（１）～（８）のいずれか１項記載の幹細胞培養用足場材料からなる樹脂膜を備える幹細胞培養用容器。
（１０）（１）～（８）のいずれか１項記載の幹細胞培養用足場材料を備える幹細胞培養用繊維。
（１１）（１）～（８）のいずれか１項記載の足場材料を用いる幹細胞の培養方法。
（１２）足場材料上に細胞塊を播種する工程を含む、（１１）記載の幹細胞の培養方法。 The present invention relates to the following contents.
(1) A scaffold material for stem cell culture, which has a dispersion component γ d of surface free energy of 24.5 or more and less than 45.0 and a dipole component γ p of surface free energy of 1.0 or more and less than 20.0.
(2) The scaffold material for stem cell culture according to (1), wherein the scaffold material for stem cell culture comprises a synthetic resin.
(3) The scaffold material for stem cell culture according to (2), wherein the synthetic resin contains at least one of a polyvinyl acetal skeleton and a poly (meth) acrylic acid ester skeleton.
(4) The scaffold material for stem cell culture according to (2), wherein the synthetic resin is a polyvinyl acetal resin.
(5) A scaffold material for stem cell culture containing a synthetic resin, wherein the synthetic resin comprises a polyvinyl acetal resin, and the scaffold material for stem cell culture has a degree of acetalization of the polyvinyl acetal resin higher than 60 mol%.
(6) The stem cell of (4) or (5), wherein the polyvinyl acetal resin has at least one selected from the group consisting of a structural unit having an imine structure, a structural unit having an amino group, and a structural unit having an amide structure. Scaffold material for culture.
(7) The polyvinyl acetal resin is such that the total content of the structural unit having an imine structure, the structural unit having an amino group, and the structural unit having an amide structure is 0.1 mol% or more and 20 mol% or less 6) The scaffold for stem cell culture described above.
(8) The scaffold material for stem cell culture according to any one of (1) to (7), wherein the stem cells are pluripotent stem cells.
(9) A container for stem cell culture comprising a resin membrane comprising the scaffold material for stem cell culture according to any one of (1) to (8) in at least a part of a cell culture region.
(10) A stem cell culture fiber comprising the scaffold material for stem cell culture according to any one of (1) to (8).
(11) A method for culturing stem cells using the scaffold material according to any one of (1) to (8).
(12) The method for culturing stem cells according to (11), which comprises the step of seeding a cell mass on a scaffold material.
　本発明によれば、適度な親水性と強度を備え、幹細胞の播種後の定着性が高い幹細胞培養用足場材料及びそれを用いた幹細胞培養方法が提供される。 According to the present invention, there is provided a scaffold material for stem cell culture which has appropriate hydrophilicity and strength, and which has high fixation after seeding of stem cells, and a stem cell culture method using the same.
図１は主な合成樹脂のγｄに対するγｐの関係をまとめた図である。FIG. 1 is a diagram summarizing the relationship of γ p to γ d of the main synthetic resin. 図２は図１の一部拡大図である。FIG. 2 is a partially enlarged view of FIG. 図３は図１の一部拡大図である。FIG. 3 is a partially enlarged view of FIG. 図４は細胞播種後２４時間後の初期接着性の評価基準を示す図である。FIG. 4 is a diagram showing the evaluation criteria of initial adhesion 24 hours after cell seeding. 図５は実施例に係る幹細胞培養用足場材料における、細胞の播種後２４時間後の位相差顕微鏡写真である。FIG. 5 is a phase contrast micrograph of a scaffold for stem cell culture according to an example 24 hours after seeding of cells. 図６は比較例に係る幹細胞培養用足場材料における、細胞の播種後２４時間後の位相差顕微鏡写真である。FIG. 6 is a phase contrast micrograph of a scaffold for stem cell culture according to a comparative example 24 hours after seeding of cells. 図７は細胞播種後５日後の細胞増殖性の評価基準を示す図である。FIG. 7 shows the evaluation criteria of cell proliferation 5 days after cell seeding. 図８は実施例に係る幹細胞培養用足場材料における、細胞の播種後５日後の位相差顕微鏡写真である。FIG. 8 is a phase contrast photomicrograph five days after seeding of cells in the scaffold for stem cell culture according to the example. 図９は比較例に係る幹細胞培養用足場材料における、細胞の播種後５日後の位相差顕微鏡写真である。FIG. 9 is a phase contrast micrograph of a scaffold for stem cell culture according to a comparative example, five days after seeding of cells.
　以下に、実施形態を挙げて本発明の説明を行うが、本発明は以下の実施形態に限定されるものではない。
　なお、本明細書で用いられる用語の説明を行う。
　「幹細胞」とは、自己複製能と分化能を有する細胞をいう。幹細胞のうち、自己複製能を有し、かつ、１つの細胞から、内胚葉、中胚葉、外胚葉の全ての細胞へ分化できるものを「多能性幹細胞」という。
　多能性幹細胞としては、例えば、人工多能性幹細胞（induced pluripotent stem cell、以下「ｉＰＳ細胞」という。）、胚性幹細胞（embryonic stem cells、以下「ＥＳ細胞」という。）、Ｍｕｓｅ細胞（multilinege differentiating stress enduring cells）、胚性がん細胞（embryonic germ cell）、胚性生殖幹細胞（embryonic germ cell）、ｍＧＳ細胞（multipotent germ stem cell）等が挙げられる。
　幹細胞のうち、自己複製能を有し、外胚葉系組織、内胚葉系組織、中胚葉系組織、生殖系組織のいずれかに属し、それが属している臓器の構成細胞種への限られた分化能を示すものを「組織幹細胞」および「組織前駆細胞」という。
　組織幹細胞および組織前駆細胞としては、例えば、神経幹細胞、神経堤幹細胞、網膜幹細胞、角膜幹細胞、ケラチノサイト表皮幹細胞、メラノサイト幹細胞、乳腺幹細胞、肝幹細胞、腸幹細胞、気道幹細胞、造血幹細胞、間葉系幹細胞、心臓幹細胞、血管内皮前駆細胞、血管周皮細胞、骨格筋幹細胞、脂肪幹細胞、腎前駆細胞、精子幹細胞等が挙げられる。この様な幹細胞としては、例えば「もっとよくわかる！幹細胞と再生医療」（羊土社、長船健二著）に記載の幹細胞を挙げることができる。 Hereinafter, the present invention will be described by way of embodiments, but the present invention is not limited to the following embodiments.
In addition, explanations of terms used in the present specification will be made.
"Stem cells" refer to cells having self-replication ability and differentiation ability. Among stem cells, those capable of self-replication and capable of differentiating from one cell to all cells of endoderm, mesoderm and ectoderm are referred to as "pluripotent stem cells".
As pluripotent stem cells, for example, induced pluripotent stem cells (hereinafter referred to as "iPS cells"), embryonic stem cells (hereinafter referred to as "ES cells"), Muse cells (multilinege) Differentiating stress enduring cells), embryonic cancer cells (embryonic germ cells), embryonic germ stem cells (embryonic germ cells), mGS cells (multipotent germ stem cells) and the like can be mentioned.
Among stem cells, they are capable of self-replication and belong to ectodermal tissue, endodermal tissue, mesodermal tissue, or germline tissue, and are restricted to constituent cell types of the organ to which they belong Those showing differentiation ability are referred to as "tissue stem cells" and "tissue precursor cells".
Tissue stem cells and tissue precursor cells include, for example, neural stem cells, neural crest stem cells, retinal stem cells, corneal stem cells, keratinocyte epidermal stem cells, melanocyte stem cells, mammary stem cells, hepatic stem cells, enteric stem cells, airway stem cells, hematopoietic stem cells, mesenchymal stem cells Cardiac stem cells, vascular endothelial progenitor cells, pericytes of vascular blood, skeletal muscle stem cells, adipose stem cells, renal progenitor cells, sperm stem cells and the like. As such stem cells, there can be mentioned, for example, the stem cells described in “More clearly! Stem cells and regenerative medicine” (by Yodosha, Kenji Nagafune).
［幹細胞培養用足場材料１］
　本発明者らは上述の課題を解決するべく、幹細胞培養用足場材料の表面自由エネルギーを制御することで、上述の課題を解決できることを見出し、本発明を完成するに至った。即ち、本発明の第一の態様は、表面自由エネルギーの分散成分γｄと双極子成分γｐが、ある一定の範囲にある幹細胞培養用足場材料に関する。
　なお、本明細書における表面自由エネルギーの分散成分γｄ及び双極子成分γｐは、Ｋａｅｌｂｌｅ－Ｕｙ理論式を用いて測定することができる。
　ここで、Ｋａｅｌｂｌｅ－Ｕｙの理論式は、式（１）で表されるように、トータル表面自由エネルギーγが分散成分γｄと、双極子成分γｐとの和からなるとの仮定に基づく。 [Staging material 1 for stem cell culture]
In order to solve the above-mentioned subject, the present inventors find out that the above-mentioned subject can be solved by controlling the surface free energy of the scaffold material for stem cell culture, and came to complete the present invention. That is, the first aspect of the present invention, the dispersion component gamma d and the dipole component gamma p of the surface free energy, to scaffolds for stem cell culture in a certain range.
The dispersion component gamma d and the dipole component gamma p of the surface free energy in this specification can be measured using the Kaelble-Uy theoretical formula.
Here, the theoretical expression of Kaelble-Uy, as represented by the formula (1), based on the assumption of a total surface free energy gamma is distributed components gamma d, and consists of the sum of the dipole component gamma p.
Figure JPOXMLDOC01-appb-M000001
 　また、液体の表面の表面自由エネルギーをγl、固体の表面自由エネルギーをγs、接触角をθで表すと、次の式（２）が成り立つ。
Figure JPOXMLDOC01-appb-M000001
 Further, when the surface free energy of the surface of the liquid is γ 1 , the surface free energy of a solid is γ s , and the contact angle is θ, the following equation (2) holds.
Figure JPOXMLDOC01-appb-M000002
 　従って、γlの成分が既知の液体を２種類用いて（本発明では純水とジヨードメタン）、幹細胞培養用足場材料に対するそれぞれの接触角θを測定し、γs d、γs pに関する連立方程式を解くことにより、幹細胞培養用足場材料の分散成分γｄ、双極子成分γｐが求められる。
　上記純水の接触角は、足場材料に純水１μＬを着滴させ、３０秒後の液滴像を、接触角計（協和界面化学社製、ＤＭｏ－７０１）を用いて撮影することで得ることができる。また、上記ジヨードメタンの接触角は、足場材料にジヨードメタン１μＬを着滴させ、同様に３０秒後の液滴像を撮影することで得ることができる。
Figure JPOXMLDOC01-appb-M000002
 Thus, components of the gamma l are used two kinds of known liquid (pure water and diiodomethane in the present invention), measuring the respective contact angle θ with respect to the scaffold for stem cell culture, gamma s d, simultaneous equations for gamma s p The dispersion component γ d and the dipole component γ p of the scaffold for stem cell culture can be obtained by solving
The contact angle of the pure water is obtained by depositing 1 μL of pure water on the scaffold material and photographing the droplet image after 30 seconds using a contact angle meter (DMo-701, manufactured by Kyowa Interface Chemical Co., Ltd.) be able to. Further, the contact angle of diiodomethane can be obtained by depositing 1 μL of diiodomethane on the scaffold material and similarly photographing a droplet image after 30 seconds.
　上記幹細胞培養用足場材料は、上記表面自由エネルギーの分散成分γｄ、及び双極子成分γｐを好適に調整できる観点から、合成樹脂を含むことが好ましい。また、合成樹脂としては、上記表面自由エネルギーの分散成分γｄ、及び双極子成分γｐを好適に調整できる観点から、ポリビニルアセタール骨格、ポリ（メタ）アクリル酸エステル骨格の少なくともいずれか一方を含むことが好ましい。
　図１は主な合成樹脂の表面自由エネルギーの分散成分γｄに対する双極子成分γｐの関係をまとめた図である。図２、図３はそれぞれ図１の一部拡大図である。
　本発明の幹細胞培養用足場材料の表面自由エネルギーの分散成分γｄは、２４．５以上４５．０未満である。上記分散成分γｄは、２８．０以上３８．０以下がより好ましく、３２．８以上３６．０以下がさらに好ましい。
　本発明の幹細胞培養用足場材料の表面自由エネルギーの双極子成分γｐは、１．０以上２０．０未満である。上記双極子成分γｐは、１．０以上１０．０以下がより好ましく、２．５以上５．０以下がさらに好ましい。
　上記分散成分γｄおよび上記双極子成分γｐは、例えば、以下で述べる合成樹脂の骨格を適宜変更することにより制御することができる。
　上記分散成分γｄは、例えば、合成樹脂の骨格の中から、非極性官能基量を増やすことや環状構造を有する官能基を導入することで大きくすることができ、合成樹脂中のブチル基成分の量を減らすこと等により小さくすることができる。合成樹脂としては、ポリビニルアセタール骨格、ポリ（メタ）アクリル酸エステル骨格の少なくともいずれか一方を含むことが好ましい。 The scaffold material for stem cell culture preferably contains a synthetic resin from the viewpoint that the dispersion component γ d of the surface free energy and the dipole component γ p can be suitably adjusted. Further, the synthetic resin contains at least one of a polyvinyl acetal skeleton and a poly (meth) acrylic acid ester skeleton from the viewpoint that the dispersion component γ d of the surface free energy and the dipole component γ p can be suitably adjusted. Is preferred.
FIG. 1 is a diagram summarizing the relationship of the dipolar component γ p to the dispersion component γ d of the surface free energy of the main synthetic resin. 2 and 3 are each a partially enlarged view of FIG.
The dispersion component γ d of the surface free energy of the scaffold material for stem cell culture of the present invention is 24.5 or more and less than 45.0. The dispersion ingredients gamma d is more preferably 28.0 or more 38.0 or less, more preferably 32.8 or more 36.0 or less.
The dipole component γ p of the surface free energy of the scaffold material for stem cell culture of the present invention is 1.0 or more and less than 20.0. As for the said dipole component (gamma) p , 1.0 or more and 10.0 or less are more preferable, and 2.5 or more and 5.0 or less are more preferable.
The dispersion ingredients gamma d and the dipole component gamma p is, for example, can be controlled by changing the backbone of the synthetic resin described below as appropriate.
The dispersion component γ d can be increased, for example, by increasing the amount of nonpolar functional groups or introducing a functional group having a cyclic structure from the skeleton of the synthetic resin, and a butyl group component in the synthetic resin Can be reduced by reducing the amount of The synthetic resin preferably contains at least one of a polyvinyl acetal skeleton and a poly (meth) acrylic acid ester skeleton.
　双極子成分γｐは、例えば、合成樹脂の骨格の中から、極性官能基量を増やすことや、エーテル構造を含む官能基を大きくすることができ、非極性官能基であるブチル基量を増やすことで小さくすることができる。 The dipole component γ p can increase, for example, the amount of polar functional groups or the functional group including an ether structure from the skeleton of a synthetic resin and increase the amount of butyl groups which are nonpolar functional groups. Can be made smaller.
［合成樹脂］
　合成樹脂は、重合性モノマー（以下、単に「モノマー」ともいう）を重合（重縮合も含む）して得られるポリマー（以下、単に「ポリマー」ともいう）を主成分とするものをいう。上記ポリマーは一種または二種以上の重合性モノマーのコポリマーも含む。 [Synthetic resin]
The synthetic resin refers to a resin having as a main component a polymer (hereinafter, also simply referred to as a “polymer”) obtained by polymerizing (also referred to as simply “a monomer”) of a polymerizable monomer (hereinafter also referred to as “monomer”). The above polymers also include copolymers of one or more polymerizable monomers.
　上記ポリマーとしては、例えば、（不）飽和炭化水素、芳香族炭化水素、（不）飽和脂肪酸、芳香族カルボン酸、（不）飽和ケトン、芳香族ケトン、（不）飽和アルコール、芳香族アルコール、（不）飽和アミン、芳香族アミン、（不）飽和チオール、芳香族チオール、有機ケイ素化合物等の１種以上の重合性モノマーからなるポリマーが挙げられる。
　具体的な上記ポリマーとしては、例えば、ポリオレフィン、ポリエーテル、ポリビニルアルコール、ポリビニルアセタール、ポリエステル、ポリ（メタ）アクリル酸エステル、エポキシ樹脂、ポリアミド、ポリイミド、ポリウレタン、ポリカーボネート、セルロース、ポリペプチド等が挙げられる。これらのなかでも、幹細胞の定着性の観点から、ポリ（メタ）アクリル酸エステル、ポリビニルアセタールが好ましく、ポリビニルアセタールがより好ましい。
　なお、これらのポリマーは、一種類のみを用いてもよく、二種類以上を組み合わせて用いてもよい。二種類以上のポリマーを組み合わせる場合は、二種類以上のポリマーを混合して用いてもよく、二種類以上のポリマーの骨格を化学結合させたポリマーとして用いてもよい。合成樹脂として、二種類以上のポリマーを組み合わせる場合には、ポリ（メタ）アクリル酸エステルと、ポリビニルアセタールとを組み合わせることが好ましい。 Examples of the polymer include (unsaturated) hydrocarbons, aromatic hydrocarbons, (unsaturated) fatty acids, aromatic carboxylic acids, (unsaturated) ketones, aromatic ketones, (unsaturated) alcohols, aromatic alcohols, Examples thereof include polymers composed of one or more polymerizable monomers such as (un) saturated amines, aromatic amines, (unsaturated) thiols, aromatic thiols, and organosilicon compounds.
Specific examples of the above-mentioned polymer include polyolefin, polyether, polyvinyl alcohol, polyvinyl acetal, polyester, poly (meth) acrylic acid ester, epoxy resin, polyamide, polyimide, polyurethane, polycarbonate, cellulose, polypeptide and the like. . Among these, poly (meth) acrylic acid esters and polyvinyl acetals are preferable, and polyvinyl acetals are more preferable, from the viewpoint of fixation of stem cells.
These polymers may be used alone or in combination of two or more. When two or more types of polymers are combined, two or more types of polymers may be mixed and used, or may be used as a polymer in which the backbones of two or more types of polymers are chemically bonded. When two or more types of polymers are combined as a synthetic resin, it is preferable to combine poly (meth) acrylic acid ester and polyvinyl acetal.
　本明細書における「（メタ）アクリル酸類」とは、（メタ）アクリル酸エステル及び（メタ）アクリル酸からなる群より選択される少なくとも１種をいう。またポリ（メタ）アクリル酸類は、そのモノマーである（メタ）アクリル酸エステル又は（メタ）アクリル酸を重合することによって得られる重合体であるが、（メタ）アクリル酸エステル又は（メタ）アクリル酸以外のモノマーを共重合したものも含む。
　上記（メタ）アクリル酸エステルとしては特に限定されないが、（メタ）アクリル酸アルキルエステル、（メタ）アクリル酸環状アルキルエステル、（メタ）アクリル酸アリールエステル、（メタ）アクリルアミド類、（メタ）アクリル酸ポリエチレングリコール類、（メタ）アクリル酸ホスホリルコリン等が挙げられる。 In the present specification, “(meth) acrylic acids” refers to at least one selected from the group consisting of (meth) acrylic esters and (meth) acrylic acids. Poly (meth) acrylic acids are polymers obtained by polymerizing (meth) acrylic acid ester or (meth) acrylic acid which is the monomer, but (meth) acrylic acid ester or (meth) acrylic acid The thing which copolymerized other monomers is also included.
The (meth) acrylic acid ester is not particularly limited, but (meth) acrylic acid alkyl ester, (meth) acrylic acid cyclic alkyl ester, (meth) acrylic acid aryl ester, (meth) acrylamides, (meth) acrylic acid Polyethylene glycols, phosphoryl choline (meth) acrylate and the like can be mentioned.
　上記（メタ）アクリル酸アルキルエステルとしては、例えば、メチル（メタ）アクリレート、エチル（メタ）アクリレート、ｎ－プロピル（メタ）アクリレート、イソプロピル（メタ）アクリレート、ｎ－ブチル（メタ）アクリレート、イソブチル（メタ）アクリレート、ｔ－ブチル（メタ）アクリレート、ｎ－オクチル（メタ）アクリレート、イソオクチル（メタ）アクリレート、２－エチルヘキシル（メタ）アクリレート、ノニル（メタ）アクリレート、イソノニル（メタ）アクリレート、デシル（メタ）アクリレート、イソデシル（メタ）アクリレート、ラウリル（メタ）アクリレート、ステアリル（メタ）アクリレート、イソテトラデシル（メタ）アクリレート等が挙げられる。
　なお、これらの（メタ）アクリル酸アルキルエステルは、特に制限はないが、炭素原子数１～３のアルコキシ基、テトラヒドロフルフリル基等の種々の置換基で置換されていてもよい。例としては、メトキシエチルアクリレート、テトラヒドロフルフリルアクリレート等が挙げられる。
　上記（メタ）アクリル酸環状アルキルエステルとしては、例えば、シクロヘキシル（メタ）アクリレート、イソボルニル（メタ）アクリレートなどが挙げられる。
　上記（メタ）アクリル酸アリールエステルとしては、例えば、フェニル（メタ）アクリレート、ベンジル（メタ）アクリレート等が挙げられる。
　上記アクリルアミド類としては、例えば、（メタ）アクリルアミド、Ｎ－イソプロピル（メタ）アクリルアミド、Ｎ－ｔｅｒｔ－ブチル（メタ）アクリルアミド、Ｎ，Ｎ’－ジメチル（メタ）アクリルアミド、(３－（メタ）アクリルアミドプロピル)トリメチルアンモニウムクロリド、４－（メタ）アクリロイルモルホリン、３－（メタ）アクリロイル－２－オキサゾリジノン、 Ｎ－[３－(ジメチルアミノ)プロピル]（メタ）アクリルアミド、Ｎ－(２－ヒドロキシエチル)（メタ）アクリルアミド、Ｎ－メチロール（メタ）アクリルアミド、６－（メタ）アクリルアミドヘキサン酸等が挙げられる。
　上記（メタ）アクリル酸ポリエチレングリコール類としては、例えば、メトキシ-ポリエチレングリコール（メタ）アクリレート、エトキシ-ポリエチレングリコール（メタ）アクリレート、ヒドロキシ-ポリエチレングリコール（メタ）アクリレート、メトキシ-ジエチレングリコール（メタ）アクリレート、エトキシ-ジエチレングリコール（メタ）アクリレート、ヒドロキシ-ジエチレングリコール（メタ）アクリレート、メトキシ-トリエチレングルコール（メタ）アクリレート、エトキシ-トリエチレングルコール（メタ）アクリレート、ヒドロキシ-トリエチレングルコール（メタ）アクリレート等が挙げられる。
　上記（メタ）アクリル酸ホスホリルコリンとしては、例えば、２－（メタ）アクリロイルオキシエチルホスホリルコリン等が挙げられる。
　（メタ）アクリル酸エステル以外のモノマーとしては、特に限定はなく、（メタ）アクリル酸、エチレン、ビニルエステル等が挙げられる。
　上記（メタ）アクリル酸エステルは単独で用いてもよく、２種以上を併用してもよい。なお、本明細書において、上記（メタ）アクリル酸とは、アクリル酸及びメタクリル酸を総称するものであり、上記（メタ）アクリレートとは、アクリレート及びメタクリレートを総称するものとする。
　なお、本発明の第一の態様は、以下で述べる第二の態様を組み合わせたものが、幹細胞の定着性を高める観点より好ましい。 Examples of the (meth) acrylic acid alkyl ester include methyl (meth) acrylate, ethyl (meth) acrylate, n-propyl (meth) acrylate, isopropyl (meth) acrylate, n-butyl (meth) acrylate, isobutyl (meth) ) Acrylates, t-Butyl (meth) acrylates, n-octyl (meth) acrylates, isooctyl (meth) acrylates, 2-ethylhexyl (meth) acrylates, nonyl (meth) acrylates, isononyl (meth) acrylates, decyl (meth) acrylates And isodecyl (meth) acrylate, lauryl (meth) acrylate, stearyl (meth) acrylate, isotetradecyl (meth) acrylate and the like.
The (meth) acrylic acid alkyl ester is not particularly limited, but may be substituted by various substituents such as an alkoxy group having 1 to 3 carbon atoms and a tetrahydrofurfuryl group. Examples include methoxyethyl acrylate, tetrahydrofurfuryl acrylate and the like.
Examples of the (meth) acrylic acid cyclic alkyl ester include cyclohexyl (meth) acrylate and isobornyl (meth) acrylate.
Examples of the (meth) acrylic acid aryl ester include phenyl (meth) acrylate, benzyl (meth) acrylate and the like.
Examples of the acrylamides include (meth) acrylamide, N-isopropyl (meth) acrylamide, N-tert-butyl (meth) acrylamide, N, N'-dimethyl (meth) acrylamide, (3- (meth) acrylamide propyl ) Trimethylammonium chloride, 4- (meth) acryloyl morpholine, 3- (meth) acryloyl-2-oxazolidinone, N- [3- (dimethylamino) propyl] (meth) acrylamide, N- (2-hydroxyethyl) (meth) And the like) acrylamide, N-methylol (meth) acrylamide, 6- (meth) acrylamide hexanoic acid and the like.
Examples of the (meth) acrylic acid polyethylene glycols include methoxy-polyethylene glycol (meth) acrylate, ethoxy-polyethylene glycol (meth) acrylate, hydroxy-polyethylene glycol (meth) acrylate, methoxy-diethylene glycol (meth) acrylate, and ethoxy -Diethylene glycol (meth) acrylate, hydroxy-diethylene glycol (meth) acrylate, methoxy-triethylene glycol (meth) acrylate, ethoxy-triethylene glycol (meth) acrylate, hydroxy-triethylene glycol (meth) acrylate etc. Be
Examples of the (meth) acrylic acid phosphoryl choline include 2- (meth) acryloyl oxyethyl phosphoryl choline and the like.
There is no limitation in particular as monomers other than (meth) acrylic acid ester, (meth) acrylic acid, ethylene, vinyl ester etc. are mentioned.
The (meth) acrylic acid esters may be used alone or in combination of two or more. In the present specification, the above (meth) acrylic acid is a generic term of acrylic acid and methacrylic acid, and the above (meth) acrylate is a generic term of acrylate and methacrylate.
The first aspect of the present invention is preferably a combination of the second aspects described below from the viewpoint of enhancing the fixability of stem cells.
［幹細胞培養用足場材料２］
　本発明者らは、鋭意検討した結果、ポリビニルアセタール樹脂を含有する合成樹脂を用いることで、上述の課題を解決できることを見出し、本発明を完成するに至った。
　本発明の第二の態様は、合成樹脂を含有する幹細胞培養用足場材料であって、合成樹脂がポリビニルアセタール樹脂を含み、ポリビニルアセタール樹脂のアセタール化度が６０モル％よりも高い幹細胞培養用足場材料に関する。なお、本発明の幹細胞培養用足場材料には、合成樹脂のみからなる態様が含まれる。
　この幹細胞培養用足場材料は、適度な親水性と強度を備えるため、幹細胞の播種後の定着性が向上する。特にフィーダー細胞や接着タンパク質を含まない無血清培地培養において、幹細胞播種後の初期定着率が向上する。 [Staging material 2 for stem cell culture]
As a result of intensive studies, the present inventors have found that the above-mentioned problems can be solved by using a synthetic resin containing a polyvinyl acetal resin, and the present invention has been accomplished.
The second aspect of the present invention is a scaffold material for stem cell culture containing a synthetic resin, wherein the synthetic resin comprises a polyvinyl acetal resin, and the scaffold for stem cell culture has a degree of acetalization of polyvinyl acetal resin higher than 60 mol%. It relates to the material. The scaffold material for stem cell culture of the present invention includes an embodiment comprising only a synthetic resin.
Since the scaffold material for stem cell culture has appropriate hydrophilicity and strength, the fixation after seeding of stem cells is improved. In particular, in a serum-free medium culture that does not contain feeder cells or adhesion proteins, the initial retention rate after stem cell seeding is improved.
　従来、幹細胞培養用足場材料として合成樹脂を用いる場合に、合成樹脂のアセタール化度を６０モル％よりも高く設定することは報告されていなかった。アセタール化度の増加に伴う水酸基の割合の低下により樹脂の親水性が低下することで、幹細胞培養用足場材料への幹細胞の播種後の定着性の低下や、または細胞培養に必要な多糖類等の透過性の低下が懸念されていたからである。ところが、本発明者等は、親水性よりも強度が重要であることを知見し、アセタール化度を６０モル％よりも高く設定して幹細胞培養用足場材料の強度を向上することで、幹細胞の播種後の定着性が向上することを見出し、本発明を完成するに至った。以下、ポリビニルアセタール樹脂について詳細に説明する。 Heretofore, in the case of using a synthetic resin as a scaffold material for stem cell culture, setting the degree of acetalization of the synthetic resin to be higher than 60 mol% has not been reported. Since the hydrophilicity of the resin is reduced due to the decrease in the proportion of hydroxyl groups with the increase in the degree of acetalization, the fixability after stem cell seeding to the scaffold material for stem cell culture decreases, or polysaccharides etc. necessary for cell culture Because of the concern about the decrease in However, the present inventors have found that strength is more important than hydrophilicity, and by setting the degree of acetalization to be higher than 60 mol% to improve the strength of the scaffold material for stem cell culture, stem cells It has been found that the fixability after seeding is improved, and the present invention has been completed. Hereinafter, polyvinyl acetal resin is demonstrated in detail.
（ポリビニルアセタール樹脂）
　ポリビニルアセタール樹脂は、ポリビニルアルコールをアルデヒドによりアセタール化することにより合成される樹脂であり、側鎖にアセチル基と水酸基、そしてアセタール基を有する。 (Polyvinyl acetal resin)
The polyvinyl acetal resin is a resin synthesized by acetalizing polyvinyl alcohol with an aldehyde, and has an acetyl group, a hydroxyl group and an acetal group in the side chain.
　上記ポリビニルアセタール樹脂のアセタール化度は、好ましい下限が６０モル％、好ましい上限は９０モル％である。アセタール化度が６０モル％以上であると、幹細胞の定着性に優れ、高効率に細胞増殖を行うことができる。また、アセタール化度が９０モル％以下であると、溶剤への溶解性を良好なものとすることができる。より好ましい下限が６５モル％、より好ましい上限が８５モル％である。
　上記ポリビニルアセタール樹脂のアセタール度は１Ｈ-ＮＭＲ測定により測定可能である。 The lower limit of the degree of acetalization of the polyvinyl acetal resin is preferably 60 mol%, and more preferably 90 mol%. When the degree of acetalization is 60% by mole or more, stem cell fixability is excellent and cell proliferation can be performed with high efficiency. Moreover, the solubility to a solvent can be made favorable as the degree of acetalization is 90 mol% or less. A more preferable lower limit is 65 mol%, and a more preferable upper limit is 85 mol%.
The degree of acetal of the polyvinyl acetal resin can be measured by 1 H-NMR measurement.
　上記アセタール化に用いられるアルデヒドとしては、炭素数１～１０の鎖状脂肪族基、環状脂肪族基又は芳香族基を有するアルデヒドが挙げられる。これらのアルデヒドとしては、従来公知のアルデヒドを使用できる。
　上記アルデヒドの種類は特に限定されないが、ホルムアルデヒド、アセトアルデヒド、プロピオンアルデヒド、ブチルアルデヒド、ペンタナール、ヘキサナール、ヘプタナール、オクタナール、ノナナール、デカナール、アクロレイン、ベンズアルデヒド、シンナムアルデヒド、ペリルアルデヒド、ホルミルピリジン、ホルミルイミダゾール、ホルミルピロール、ホルミルピペリジン、ホルミルピペリジン、ホルミルトリアゾール、ホルミルテトラゾール、ホルミルインドール、ホルミルイソインドール、ホルミルプリン、ホルミルプリン、ホルミルベンゾイミダゾール、ホルミルベンゾトリアゾール、ホルミルキノリン、ホルミルイソキノリン、ホルミルキノキサリン、ホルミルシンノリン、ホルミルプテリジン、ホルミルフラン、ホルミルオキソラン、ホルミルオキサン、ホルミルチオフェン、ホルミルチオラン、ホルミルチアン、ホルミルアデニン、ホルミルグアニン、ホルミルシトシン、ホルミルチミン、ホルミルウラシルなどが挙げられる。上記アルデヒドは鎖状であっても環状であっても良い。 Examples of the aldehyde used for the acetalization include aldehydes having a linear aliphatic group having 1 to 10 carbon atoms, a cyclic aliphatic group or an aromatic group. As these aldehydes, conventionally known aldehydes can be used.
The type of aldehyde is not particularly limited, but formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, pentanal, hexanal, heptanal, octanal, nonanal, decanal, acrolein, benzaldehyde, cinnamaldehyde, perylaldehyde, formyl pyridine, formyl imidazole, formyl pyrrole Formylpiperidine, formylpiperidine, formyltriazole, formyltetrazole, formylindole, formyl isoindole, formylpurine, formylpurine, formylbenzimidazole, formylbenzotriazole, formylquinoline, formyl isoquinoline, formylquinoxaline, formylcinnoline, formylpteridine, Formylfuran, Holmy Oxolane, Horumiruokisan, formyl thiophene, Horumiruchioran, Horumiruchian, formyl adenine, formyl guanine, formyl cytosine, Horumiruchimin, like formyl uracil. The aldehyde may be linear or cyclic.
　上記アルデヒドはホルムアルデヒド、アセトアルデヒド、プロピオンアルデヒド、ブチルアルデヒド、ペンタナールであることが好ましく、ブチルアルデヒドがさらに好ましい。 The aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde or pentanal, and more preferably butyraldehyde.
　ポリビニルアセタール樹脂の重合度の下限は、１００が好ましく、２００がより好ましく、５００がさらに好ましく、１５００がさらにより好ましい。重合度が上記範囲であると、細胞培養にしようする培地で膨潤しても足場材強度を好適に保つことが出来ることから、細胞増殖性が向上する。重合度の上限は６０００が好ましく、３０００がより好ましく、２５００がさらに好ましい。重合度が上記範囲であると、取り扱い性が良く、足場材を好適に成形出来る。 The lower limit of the polymerization degree of the polyvinyl acetal resin is preferably 100, more preferably 200, still more preferably 500, and still more preferably 1500. When the degree of polymerization is in the above-mentioned range, the scaffold strength can be suitably maintained even if it is swollen with a culture medium used for cell culture, whereby cell proliferation is improved. The upper limit of the polymerization degree is preferably 6000, more preferably 3000, and still more preferably 2500. When the polymerization degree is in the above range, the handling property is good, and the scaffold can be suitably formed.
　上記ポリビニルアルコールは、ビニル化合物との共重合体であっても良い。ビニル化合物としては、エチレン、アリルアミン、ビニルピロリドン、無水マレイン酸、マレイミド、イタコン酸、（メタ）アクリル酸、ビニルアミン、（メタ）アクリル酸エステル等が挙げられる。なお、上記（メタ）アクリル酸エステルとしては、例えば、上述した（メタ）アクリル酸エステルを用いることができる。 The polyvinyl alcohol may be a copolymer with a vinyl compound. Examples of the vinyl compound include ethylene, allylamine, vinylpyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth) acrylic acid, vinylamine, (meth) acrylic acid ester and the like. In addition, as said (meth) acrylic acid ester, the (meth) acrylic acid ester mentioned above can be used, for example.
　上記ポリビニルアセタール樹脂は、ビニル化合物とのグラフト共重合体であっても良い。ビニル化合物としては、上述の化合物が挙げられる。 The polyvinyl acetal resin may be a graft copolymer with a vinyl compound. As a vinyl compound, the above-mentioned compound is mentioned.
　上記グラフト共重合体は「ポリビニルアセタールからなるユニット」と「ビニル化合物からなるユニット」とを有するグラフト共重合体（以下、単に「グラフト共重合体」ともいう）を含有する。ビニル化合物とは、エテニル基（Ｈ２Ｃ＝ＣＨ－）を有する構成単位を有する化合物をいう。
　本発明において、「ポリビニルアセタールからなるユニット」及び「ビニル化合物からなるユニット」とは、グラフト共重合体中に存在している「ポリビニルアセタール」、「ビニル化合物からなるユニット」のことをいう。また、ポリビニルアセタールからなるユニット及びビニル化合物からなるユニットからなるユニットを有するグラフト共重合体は、主鎖を構成する「ポリビニルアセタールからなるユニット」又は「ビニル化合物からなるユニット」に、該主鎖とは異なる側鎖を構成する「ポリビニルアセタールからなるユニット」又は「ビニル化合物からなるユニット」が結合した分岐状の共重合体のことをいう。 The graft copolymer contains a graft copolymer (hereinafter, also simply referred to as "graft copolymer") having "a unit composed of a polyvinyl acetal" and a "unit composed of a vinyl compound". The vinyl compound refers to a compound having a constitutional unit having an ethenyl group (H 2 C = CH—).
In the present invention, the "unit made of polyvinyl acetal" and the "unit made of vinyl compound" refer to the "polyvinyl acetal" and the "unit made of vinyl compound" present in the graft copolymer. Further, a graft copolymer having a unit composed of a unit composed of polyvinyl acetal and a unit composed of a unit composed of a vinyl compound is a “unit composed of polyvinyl acetal” constituting a main chain or a “unit composed of a vinyl compound” The term “branched copolymer” refers to a “polyvinyl acetal unit” or “vinyl compound unit” constituting different side chains.
　上記グラフト共重合体の分子量としては特に制限は無いが、数平均分子量（Ｍｎ）が１００００～６０００００で、重量平均分子量（Ｍｗ）が２００００～１２０００００で、これらの比（Ｍｗ／Ｍｎ）が２．０～４０であることが好ましい。Ｍｎ、Ｍｗ、Ｍｗ／Ｍｎがこのような範囲であると、上記幹細胞足場材料の強度が好適に保たれる。 The molecular weight of the graft copolymer is not particularly limited, but the number average molecular weight (Mn) is 10000 to 600000, the weight average molecular weight (Mw) is 20000 to 1200000, and the ratio (Mw / Mn) thereof is 2.%. It is preferably from 0 to 40. When the Mn, Mw, and Mw / Mn are in such ranges, the strength of the stem cell scaffold material is suitably maintained.
　上記グラフト共重合体におけるアセタール化度は、例えば上記グラフト共重合体のキシレンにおける可溶分を重水素化ジメチルスルホキシドに溶解し、１Ｈ-ＮＭＲ測定によりアセタール化度を測定する方法が挙げられる。 The degree of acetalization in the above graft copolymer can be obtained, for example, by dissolving the soluble matter in xylene of the above graft copolymer in deuterated dimethyl sulfoxide and measuring the degree of acetalization by 1 H-NMR measurement.
　上記ポリビニルアセタール樹脂は、その一部にブレンステッド塩基性基またはブレンステッド酸性基を有することが好ましい。すなわち、ポリビニルアセタール樹脂の一部がブレンステッド塩基性基またはブレンステッド酸性基により変性されていることが好ましく、ポリビニルアセタール樹脂の一部がブレンステッド塩基性基で変性されていることがより好ましい。ポリビニルアセタール樹脂の一部がブレンステッド塩基性基またはブレンステッド酸性基により変性されている場合、フィーダー細胞や接着タンパク質を含まない無血清培地培養において、幹細胞播種後の初期定着率が向上し、幹細胞の培養がし易くなる。
　なお、本明細書において、ポリビニルアセタール樹脂の一部にブレンステッド塩基性基またはブレンステッド酸性基を有するポリビニルアセタール樹脂を変性ポリビニルアセタール樹脂という。 The polyvinyl acetal resin preferably has a Bronsted basic group or a Bronsted acidic group in part. That is, it is preferable that a part of the polyvinyl acetal resin is modified with a Bronsted basic group or a Bronsted acidic group, and it is more preferable that a part of the polyvinyl acetal resin is modified with a Bronsted basic group. When a part of the polyvinyl acetal resin is denatured by a Br ス テ ッ ド nsted basic group or Br ブ レ ン nsted acid group, the initial retention rate after seeding with stem cells is improved in a serum-free medium culture containing no feeder cells or adhesion proteins, and stem cells Cultivation of
In the present specification, a polyvinyl acetal resin having a Bronsted basic group or a Bronsted acidic group in part of the polyvinyl acetal resin is referred to as a modified polyvinyl acetal resin.
　上記ブレンステッド塩基性基は、水素イオンＨ＋を他の物質から受け取ることができる官能基の総称である。ブレンステッド塩基性基としては、例えば、アミン構造を有する置換基、イミン構造を有する置換基、アミド構造を有する置換基、イミド構造を有する置換基等のアミン系塩基性基が挙げられる。
　従って、このようなポリビニルアセタール樹脂としては、アミン構造を有する構成単位、イミン構造を有する構成単位、アミド構造を有する構成単位、及びイミド構造を有する構成単位からなる群から選択される少なくとも一種を構成単位として含むポリビニルアセタール樹脂が好ましい。上記アミン構造を有する構成単位、イミン構造を有する構成単位、アミド構造を有する構成単位、及びイミド構造を有する構成単位の合計の含有量は、ポリビニルアセタール樹脂中、０．１モル％～３０モル％であることが好ましく、播種直後の細胞接着性の観点から、１モル％～１０モル％であることがより好ましい。 The Br ス テ ッ ド nsted basic group is a generic term for functional groups that can receive hydrogen ion H + from other substances. Examples of the Br ス テ ッ ド nsted basic group include amine basic groups such as a substituent having an amine structure, a substituent having an imine structure, a substituent having an amide structure, and a substituent having an imide structure.
Therefore, as such a polyvinyl acetal resin, at least one selected from the group consisting of a structural unit having an amine structure, a structural unit having an imine structure, a structural unit having an amide structure, and a structural unit having an imide structure The polyvinyl acetal resin contained as a unit is preferable. The total content of the structural unit having an amine structure, the structural unit having an imine structure, the structural unit having an amide structure, and the structural unit having an imide structure is 0.1 mol% to 30 mol% in the polyvinyl acetal resin It is preferably from 1 mol% to 10 mol% from the viewpoint of cell adhesion immediately after seeding.
　本発明において、上記イミン構造とは、Ｃ＝Ｎ結合を有する構造をいう。上記ポリビニルアセタール樹脂は、イミン構造を側鎖に有することが好ましい。また、上記イミン構造は、ポリビニルアセタール樹脂の主鎖を構成する炭素に直接結合してもよく、アルキレン基等の連結基を介して結合していてもよい。なお、上記イミン構造を側鎖に有するとは、上記イミン構造をポリビニルアセタール樹脂のグラフト鎖に有することを含む。上記イミン構造を有する構成単位としては、例えば、下記式（１）に示す構成単位が挙げられる。 In the present invention, the imine structure refers to a structure having a C = N bond. It is preferable that the said polyvinyl acetal resin has an imine structure in a side chain. The imine structure may be directly bonded to carbon constituting the main chain of the polyvinyl acetal resin, or may be bonded via a linking group such as an alkylene group. In addition, having an imine structure in a side chain includes having the imine structure in a graft chain of a polyvinyl acetal resin. As a structural unit which has the said imine structure, the structural unit shown to following formula (1) is mentioned, for example.
Figure JPOXMLDOC01-appb-C000003
 　式（１）中、Ｒ１は、単結合、又は、アルキレン基を表し、Ｒ２は、イミン構造を有する基を表す。
Figure JPOXMLDOC01-appb-C000003
 In formula (1), R 1 represents a single bond or an alkylene group, and R 2 represents a group having an imine structure.
　上記式（１）中、Ｒ１がアルキレン基である場合、該アルキレン基の炭素数の好ましい下限は１、好ましい上限は１２である。上記アルキレン基の炭素数が１２を超えると、最適な強度が得られないことがある。上記Ｒ１がアルキレン基である場合、上記アルキレン基の炭素数のより好ましい上限は５である。 In the above formula (1), when R 1 is an alkylene group, the preferable lower limit of the carbon number of the alkylene group is 1, and the preferable upper limit is 12. When the carbon number of the above-mentioned alkylene group exceeds 12, optimum strength may not be obtained. When said R < 1 > is an alkylene group, the more preferable upper limit of carbon number of the said alkylene group is five.
　上記式（１）中、Ｒ１がアルキレン基である場合、該アルキレン基としては、例えば、メチレン基、エチレン基、トリメチレン基、テトラメチレン基、ペンタメチレン基、ヘキサメチレン基、オクタメチレン基、デカメチレン基等の直鎖状アルキレン基、メチルメチレン基、メチルエチレン基、１－メチルペンチレン基、１，４－ジメチルブチレン基等の分岐状アルキレン基、シクロプロピレン基、シクロブチレン基、シクロヘキシレン基等の環状アルキレン基等が挙げられる。なかでも、メチレン基、エチレン基、トリメチレン基、テトラメチレン基等の直鎖状アルキル基が好ましく、メチレン基、エチレン基がより好ましい。 In the above formula (1), when R 1 is an alkylene group, examples of the alkylene group include methylene group, ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, octamethylene group, decamethylene group. , Linear alkylene groups such as methylmethylene group, methylethylene group, 1-methylpentylene group, branched alkylene group such as 1,4-dimethylbutylene group, cyclopropylene group, cyclobutylene group, cyclohexylene group, etc. And cyclic alkylene groups of the following. Among them, linear alkyl groups such as methylene, ethylene, trimethylene and tetramethylene are preferable, and methylene and ethylene are more preferable.
　上記Ｒ２としては、下記式（２）に示す官能基が挙げられる。 As the R 2, it includes a functional group represented by the following formula (2).
Figure JPOXMLDOC01-appb-C000004
 　式（２）中、Ｒ３は水素原子又は炭素数１～１８の炭化水素基を表し、Ｒ４は炭素数１～１８の炭化水素基を表す。
Figure JPOXMLDOC01-appb-C000004
 In formula (2), R 3 represents a hydrogen atom or a hydrocarbon group having 1 to 18 carbon atoms, and R 4 represents a hydrocarbon group having 1 to 18 carbon atoms.
　上記炭化水素基としては、飽和炭化水素基、不飽和炭化水素基、芳香族系炭化水素基等が挙げられる。なお、上記炭化水素基は、飽和炭化水素基、不飽和炭化水素基、芳香族系炭化水素基のいずれか一種のみからなるものであってもよく、これらが２種以上用いられたものであってもよい。 As said hydrocarbon group, a saturated hydrocarbon group, an unsaturated hydrocarbon group, an aromatic hydrocarbon group etc. are mentioned. The above-mentioned hydrocarbon group may consist of only one kind of a saturated hydrocarbon group, an unsaturated hydrocarbon group, and an aromatic hydrocarbon group, and two or more kinds of these are used. May be
　上記飽和炭化水素基としては、例えば、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｎ－ブチル基、ｉｓｏ－ブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、ペンチル基、へキシル基、へプチル基、２－エチルヘキシル基、オクチル基、ノニル基、デシル基、ウンデシル基、ドデシル基、トリデシル基、テトラデシル基、ペンタデシル基、オクタデシル基等が挙げられる。なかでも、メチル基、エチル基、ｎ－プロピル基、ｎ－ブチル基が好ましい。
　上記芳香族系炭化水素基としては、例えば、フェニル基、トルイル基、キシリル基、ｔ－ブチルフェニル基、ベンジル基等が挙げられる。 Examples of the saturated hydrocarbon group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, iso-butyl group, sec-butyl group, tert-butyl group, pentyl group and hexyl group. And heptyl group, 2-ethylhexyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, octadecyl group and the like. Among them, methyl group, ethyl group, n-propyl group and n-butyl group are preferable.
Examples of the aromatic hydrocarbon group include phenyl group, toluyl group, xylyl group, t-butylphenyl group, benzyl group and the like.
　上記変性ポリビニルアセタール樹脂において、上記イミン構造を有する構成単位中、Ｒ１が単結合、Ｒ３が水素原子、メチル基又はエチル基、Ｒ４がメチル基、又はエチル基又はプロピル基であることが好ましい。 In the modified polyvinyl acetal resin, it is preferable that R 1 is a single bond, R 3 is a hydrogen atom, a methyl group or an ethyl group, and R 4 is a methyl group, an ethyl group or a propyl group in structural units having the imine structure. .
　上記ポリビニルアセタール樹脂は、イミン構造を有する構成単位の含有量の好ましい下限が０．１モル％、好ましい上限が２０．０モル％である。上記イミン構造を有する構成単位の含有量が０．１モル％以上であると、経時粘度安定性が良好なものとなる。上記イミン構造を有する構成単位の含有量が２０．０モル％以下であると、アセタール化を充分に進行させることができる。上記イミン構造を有する構成単位の含有量のより好ましい下限は１．０モル％、より好ましい上限は１５．０モル％である。
　なお、上記イミン構造を有する構成単位の含有量は、１Ｈ-ＮＭＲ測定により測定することができる。 As for the said polyvinyl acetal resin, the preferable lower limit of content of the structural unit which has an imine structure is 0.1 mol%, and a preferable upper limit is 20.0 mol%. When the content of the structural unit having an imine structure is 0.1 mol% or more, the viscosity stability over time becomes good. Acetalization can fully be advanced as content of the structural unit which has the said imine structure is 20.0 mol% or less. The more preferable lower limit of the content of the constituent unit having an imine structure is 1.0 mol%, and the more preferable upper limit is 15.0 mol%.
The content of the constituent unit having the imine structure can be measured by 1 H-NMR measurement.
　上記ポリビニルアセタール樹脂において、イミン構造を有する構成単位の含有量と、後述するアセタール化度の比率（イミン構造を有する構成単位の含有量／アセタール化度）は、０．００１～０．５が好ましい。上記範囲内とすることで、高い強度及び優れた接着性を両立して、接着後の耐久性を向上させることが可能となる。 In the polyvinyl acetal resin, the ratio of the content of the structural unit having an imine structure to the degree of acetalization described later (the content of the structural unit having an imine structure / the degree of acetalization) is preferably 0.001 to 0.5. . By setting it in the said range, it becomes possible to make high intensity | strength and the outstanding adhesiveness make compatible, and to improve the durability after adhesion | attachment.
　上記ポリビニルアセタール樹脂は、イミノ基（＝ＮＨ）構造を有する構成単位を有することが好ましい。
　上記ポリビニルアセタール樹脂は、上記イミノ基を側鎖に有することが好ましい。また、上記イミノ基は、ポリビニルアセタール樹脂の主鎖を構成する炭素に直接結合してもよく、アルキレン基等の連結基を介して結合していてもよい。 The polyvinyl acetal resin preferably has a structural unit having an imino group (= NH) structure.
It is preferable that the said polyvinyl acetal resin has the said imino group in a side chain. The imino group may be directly bonded to carbon constituting the main chain of the polyvinyl acetal resin, or may be bonded via a linking group such as an alkylene group.
　上記変性ポリビニルアセタール樹脂は、アミン構造を有する構成単位又はアミド構造を有する構成単位を有することが好ましい。
　上記変性ポリビニルアセタール樹脂は、上記アミン構造又はアミド構造を側鎖に有することが好ましい。また、上記アミン構造又はアミド構造は、変性ポリビニルアセタール樹脂の主鎖を構成する炭素に直接結合してもよく、アルキレン基等の連結基を介して結合していてもよい。更に、上記アミン構造は第一級アミンでもよく、第二級アミンでもよく、第三級アミンでもよく、第四級アミンでもよい。これらのなかでも、幹細胞の定着性を高める観点から、第一級アミンが好ましい。
　なお、上記アミン構造又はアミド構造を側鎖に有するとは、上記アミン構造又はアミド構造を変性ポリビニルアセタール樹脂のグラフト鎖に有することを意味する。
　特に、上記アミン構造は、－ＮＨ２であることが好ましい。なお、本発明において、アミド構造とは、－Ｃ（＝Ｏ）－ＮＨ－を有する構造をいう。なかでも、上記アミン構造を有する構成単位は、下記式（３）に示す構造であることが好ましい。また、上記アミド構造を有する構成単位は、下記式（４）に示す構造であることが好ましい。 The modified polyvinyl acetal resin preferably has a structural unit having an amine structure or a structural unit having an amide structure.
It is preferable that the said modified polyvinyl acetal resin has the said amine structure or an amide structure in a side chain. The above amine structure or amide structure may be directly bonded to carbon constituting the main chain of the modified polyvinyl acetal resin, or may be bonded via a linking group such as an alkylene group. Further, the above amine structure may be a primary amine, a secondary amine, a tertiary amine or a quaternary amine. Among these, primary amines are preferred from the viewpoint of enhancing the fixability of stem cells.
In addition, having the above-mentioned amine structure or an amide structure in a side chain means having the above-mentioned amine structure or an amide structure in the graft chain of denaturation polyvinyl acetal resin.
In particular, the amine structure is preferably -NH 2 . In the present invention, an amide structure refers to a structure having —C (= O) —NH—. Especially, it is preferable that the structural unit which has the said amine structure is a structure shown to following formula (3). Moreover, it is preferable that the structural unit which has the said amide structure is a structure shown to following formula (4).
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000006
 　式（４）中、Ｒ５は水素原子又は炭素数１～１０の炭化水素基を表す。なお、上記炭化水素基としては、アルキル基、アルケニル基、シクロアルキル基、シクロアルケニル基が挙げられる。
Figure JPOXMLDOC01-appb-C000006
 In formula (4), R 5 represents a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms. In addition, as said hydrocarbon group, an alkyl group, an alkenyl group, a cycloalkyl group, and a cycloalkenyl group are mentioned.
　上記アミン構造又はアミド構造を有する構成単位の含有量の好ましい下限は０．１モル％、好ましい上限は２０モル％である。上記アミン構造又はアミド構造を有する構成単位の含有量が０．１モル％以上であると、付加特性を充分なものとすることができる。上記含有量が２０モル％以下であると、溶解性が上がりすぎることがなく、沈殿法による変性ポリビニルアセタール樹脂粉末の取り出しが容易となる。上記含有量のより好ましい下限は０．５モル％、より好ましい上限は１０モル％である。なお、上記アミン構造又はアミド構造を有する構成単位の含有量は１Ｈ-ＮＭＲ測定により測定可能である。また、上記アミン構造又はアミド構造を有する構成単位と、イミン構造を有する構成単位とを合計した含有量の好ましい下限は０．１モル％、好ましい上限は２０モル％である。上記含有量のより好ましい下限は０．５モル％、より好ましい上限は１０モル％である。 The preferable lower limit of the content of the constituent unit having an amine structure or an amide structure is 0.1 mol%, and the preferable upper limit is 20 mol%. When the content of the constituent unit having the amine structure or the amide structure is 0.1 mol% or more, the addition property can be made sufficient. When the content is 20 mol% or less, the solubility does not increase excessively, and the removal of the modified polyvinyl acetal resin powder by the precipitation method becomes easy. The more preferable lower limit of the content is 0.5 mol%, and the more preferable upper limit is 10 mol%. The content of the structural unit having the above amine structure or amide structure can be measured by 1 H-NMR measurement. Moreover, the preferable lower limit of the content which totaled the structural unit which has the said amine structure or an amide structure, and the structural unit which has an imine structure is 0.1 mol%, and a preferable upper limit is 20 mol%. The more preferable lower limit of the content is 0.5 mol%, and the more preferable upper limit is 10 mol%.
　上記ポリビニルアセタール樹脂において、イミン構造を有する構成単位と、アミン構造又はアミド構造を有する構成単位との含有量の比率（イミン構造を有する構成単位／アミノ基又はアミド構造を有する構成単位）は、０．５／９９．５～９９．５／０．５であることが好ましい。上記比率が０．５／９９．５以上であると、経時粘度安定性を充分なものとすることができ、上記比率が９９．５／０．５以下であると、幹細胞の定着性向上の観点より架橋性能を充分に発揮することができる。上記比率のより好ましい下限は５／９５、より好ましい上限は９０／１０である。 In the polyvinyl acetal resin, the content ratio of the structural unit having an imine structure to the structural unit having an amine structure or an amide structure (structural unit having an imine structure / structural unit having an amino group or an amide structure) is 0 It is preferably 5 / 99.5 to 99.5 / 0.5. When the above ratio is 0.5 / 99.5 or more, the viscosity stability over time can be made sufficient, and when the above ratio is 99.5 / 0.5 or less, the stem cell fixability can be improved. The crosslinking performance can be sufficiently exhibited from the viewpoint. The more preferable lower limit of the above ratio is 5/95, and the more preferable upper limit is 90/10.
　上記ブレンステッド酸性基は、水素イオンＨ＋を他の物質に渡すことができる官能基の総称である。
　ブレンステッド酸性基としては、カルボキシル基、スルホン酸基、マレイン酸基、スルフィン酸基、スルフェン酸基、リン酸基、ホスホン酸基、及び、それらの塩等が挙げられる。ブレンステッド酸性基としては、なかでも、カルボキシル基が好ましい。
　上記ポリビニルアセタール樹脂を上記ブレンステッド酸性基により変性する方法としては特に限定されないが、上記ポリビニルアルコールを上記イタコン酸や（メタ）アクリル酸と共重合する方法、上記ポリビニルアルコールの側鎖にブレンステッド酸性基を導入する方法等によって得られる。 The Br ス テ ッ ド nsted acid group is a generic term for functional groups that can transfer hydrogen ion H + to other substances.
Examples of the Bronsted acidic group include a carboxyl group, a sulfonic acid group, a maleic acid group, a sulfinic acid group, a sulfenic acid group, a phosphoric acid group, a phosphonic acid group, and salts thereof. Among them, a carboxyl group is preferable as the Bronsted acidic group.
The method for modifying the polyvinyl acetal resin with the Br ス テ ッ ド nsted acidic group is not particularly limited, but a method of copolymerizing the polyvinyl alcohol with the itaconic acid or (meth) acrylic acid, Br ス テ ッ ド nsted acid on the side chain of the polyvinyl alcohol It is obtained by a method of introducing a group, etc.
　上記ポリビニルアセタール樹脂のアセタール化度は特に限定されないが、好ましい下限が６０モル％、好ましい上限は９０モル％である。アセタール化度が６０モル％以上であると、幹細胞の定着性に優れ、高効率に細胞増殖を行うことができる。また、アセタール化度が９０モル％以下であると、溶剤への溶解性を良好なものとすることができる。より好ましい下限が６５モル％、より好ましい上限が８５モル％である。上記ポリビニルアセタール樹脂のアセタール度は１Ｈ-ＮＭＲ測定により測定可能である。 The degree of acetalization of the polyvinyl acetal resin is not particularly limited, but the preferable lower limit is 60 mol%, and the preferable upper limit is 90 mol%. When the degree of acetalization is 60% by mole or more, stem cell fixability is excellent and cell proliferation can be performed with high efficiency. Moreover, the solubility to a solvent can be made favorable as the degree of acetalization is 90 mol% or less. A more preferable lower limit is 65 mol%, and a more preferable upper limit is 85 mol%. The degree of acetal of the polyvinyl acetal resin can be measured by 1 H-NMR measurement.
　上記ポリビニルアセタール樹脂は、アセチル基量は特に限定されず、好ましい下限が０．０００１モル％、好ましい上限が５モル％である。 The amount of acetyl groups in the polyvinyl acetal resin is not particularly limited, and the preferable lower limit is 0.0001% by mol, and the preferable upper limit is 5% by mol.
　上記ポリビニルアセタール樹脂を作製する方法としては、例えば、上記イミン構造を有する単量体と、酢酸ビニルとを共重合させることによって得られたポリ酢酸ビニルをケン化し得られたポリビニルアルコールを、従来公知の方法によりアセタール化する方法が挙げられる。また、アミノ基又はアミド構造を有する構成単位を有するポリビニルアルコールを、従来公知の方法によりアセタール化することでイミン構造を導入する方法を用いてもよい。アミノ基又はアミド構造を有する構成単位を有するポリビニルアルコールを後変性して得られたイミン構造を有する変性ポリビニルアルコールを、従来公知の方法によりアセタール化する方法を用いてもよい。更に、未変性のポリビニルアセタール樹脂を後変性させることでイミン構造を導入してもよい。すなわち、上記変性ポリビニルアセタール樹脂は、アミノ基又はアミド構造を有する構成単位を有するポリビニルアルコールのアセタール化物であってもよい。これらのなかでは、アミノ基又はアミド構造を有する構成単位を有するポリビニルアルコールをアセタール化してなることでイミン構造を有する変性ポリビニルアセタール樹脂を得る方法が好ましい。特に、このような方法を用いる場合、アセタール化に使用するアルデヒド、酸触媒の量を過剰に添加することでイミン構造を得ることが出来る。 As a method of producing the said polyvinyl acetal resin, the polyvinyl alcohol obtained by saponifying the polyvinyl acetate obtained by copolymerizing the monomer which has the said imine structure, and vinyl acetate is known conventionally, for example The method of acetalization by the method of is mentioned. Also, a method of introducing an imine structure by acetalizing a polyvinyl alcohol having a structural unit having an amino group or an amide structure by a conventionally known method may be used. The modified polyvinyl alcohol having an imine structure obtained by post-modifying a polyvinyl alcohol having a structural unit having an amino group or an amide structure may be acetalized by a conventionally known method. Furthermore, the imine structure may be introduced by post-denaturing the unmodified polyvinyl acetal resin. That is, the modified polyvinyl acetal resin may be an acetalized polyvinyl alcohol having a structural unit having an amino group or an amide structure. Among these, a method of obtaining a modified polyvinyl acetal resin having an imine structure by acetalizing a polyvinyl alcohol having a structural unit having an amino group or an amide structure is preferable. In particular, when such a method is used, an imine structure can be obtained by adding an excess amount of an aldehyde and an acid catalyst used for acetalization.
　上記アルデヒドを過剰に添加する方法では、アミノ基又はアミド構造を有する構成単位を有するポリビニルアルコール１００重量部に対して、アルデヒドを７０～１５０重量部添加することが好ましい。特に、アルデヒドとしては、アセトアルデヒド、プロピオンアルデヒド、ｎ－ブチルアルデヒド、イソブチルアルデヒド、ｎ－バレルアルデヒド、フェニルアルデヒドが好ましい。 In the method of adding an aldehyde in excess, it is preferable to add 70 to 150 parts by weight of aldehyde to 100 parts by weight of polyvinyl alcohol having a structural unit having an amino group or an amide structure. In particular, as the aldehyde, acetaldehyde, propionaldehyde, n-butyraldehyde, isobutyraldehyde, n-valeraldehyde and phenylaldehyde are preferable.
　上記酸触媒を過剰に添加する方法では、酸触媒を全体の０．５重量％以上添加することが好ましい。また、アミノ基又はアミド構造を有する構成単位を有するポリビニルアルコール１００重量部に対して、酸触媒を５．０～７０．０重量部添加することが好ましい。特に、酸触媒としては、塩酸、硝酸、硫酸、パラトルエンスルホン酸が好ましい。なお、このような方法を用いる場合において、アミノ基、アミド構造を有する構成単位、イミン構造を有する構成単位を確認する方法としては、例えば、１Ｈ-ＮＭＲで確認する方法等が挙げられる。 In the method of adding the acid catalyst in excess, it is preferable to add the acid catalyst by 0.5% by weight or more of the whole. Furthermore, it is preferable to add 5.0 to 70.0 parts by weight of an acid catalyst to 100 parts by weight of polyvinyl alcohol having a structural unit having an amino group or an amide structure. In particular, hydrochloric acid, nitric acid, sulfuric acid and paratoluenesulfonic acid are preferable as the acid catalyst. In the case of using such a method, examples of a method of confirming a structural unit having an amino group, an amide structure, and a structural unit having an imine structure include a method of confirming by 1 H-NMR.
　上記アセタール化は、公知の方法を用いることができ、水溶媒中、水と水との相溶性のある有機溶媒との混合溶媒中、あるいは有機溶媒中で行うことが好ましい。上記水との相溶性のある有機溶媒としては、例えば、アルコール系有機溶剤を用いることができる。上記有機溶媒としては、例えば、アルコール系有機溶剤、芳香族有機溶剤、脂肪族エステル系溶剤、ケトン系溶剤、低級パラフィン系溶剤、エーテル系溶剤、アミン系溶剤等が挙げられる。上記アルコール系有機溶剤としては、例えば、メタノール、エタノール、ｎ－プロパノール、イソプロパノール、ｎ－ブタノール、ｔｅｒｔ－ブタノール等が挙げられる。上記芳香族有機溶剤としては、例えば、キシレン、トルエン、エチルベンゼン、安息香酸メチル等が挙げられる。
　上記脂肪族エステル系溶剤としては、例えば、酢酸メチル、酢酸エチル、酢酸ブチル、プロピオン酸メチル、プロピオン酸エチル、酪酸メチル、酪酸エチル、アセト酢酸メチル、アセト酢酸エチル等が挙げられる。
　上記ケトン系溶剤としては、例えば、アセトン、メチルエチルケトン、メチルイソブチルケトン、シクロヘキサノン、メチルシクロヘキサノン、ベンゾフェノン、アセトフェノン等が挙げられる。上記低級パラフィン系溶剤としては、ヘキサン、ペンタン、オクタン、シクロヘキサン、デカン等が挙げられる。上記エーテル系溶剤としては、ジエチルエーテル、テトラヒドロフラン、エチレングリコールジメチルエーテル、エチレングリコールジエチルエーテル、プロピレングリコールジエチルエーテル等が挙げられる。上記アミド系溶剤としては、Ｎ，Ｎ－ジメチルホルムアミド、Ｎ，Ｎ－ジメチルテセトアミド、Ｎ－メチルピロリドン、アセトアニリド等が挙げられる。
上記アミン系溶剤としては、アンモニア、トリメチルアミン、トリエチルアミン、ｎ－ブチルアミン、ジｎ－ブチルアミン、トリｎ－ブチルアミン、アニリン、Ｎ－メチルアニリン、Ｎ，Ｎ－ジメチルアニリン、ピリジン等が挙げられる。
　これらは、単体で用いることもできるし、２種以上の溶媒を混合で用いることもできる。これらのなかでも、樹脂に対する溶解性及び精製時の簡易性の観点から、エタノール、ｎ－プロパノール、イソプロパノール、テトラヒドロフランが特に好ましい。 The acetalization can be carried out using a known method, and is preferably carried out in an aqueous solvent, a mixed solvent of water and an organic solvent compatible with water, or an organic solvent. As the organic solvent compatible with water, for example, an alcohol-based organic solvent can be used. Examples of the organic solvent include alcohol organic solvents, aromatic organic solvents, aliphatic ester solvents, ketone solvents, lower paraffin solvents, ether solvents, amine solvents and the like. Examples of the alcohol-based organic solvent include methanol, ethanol, n-propanol, isopropanol, n-butanol, tert-butanol and the like. Examples of the aromatic organic solvent include xylene, toluene, ethylbenzene, methyl benzoate and the like.
Examples of the aliphatic ester solvents include methyl acetate, ethyl acetate, butyl acetate, methyl propionate, ethyl propionate, methyl butyrate, ethyl butyrate, methyl acetoacetate, ethyl acetoacetate and the like.
Examples of the ketone solvents include acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, methyl cyclohexanone, benzophenone, acetophenone and the like. Examples of the lower paraffinic solvents include hexane, pentane, octane, cyclohexane and decane. Examples of the ether solvents include diethyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, propylene glycol diethyl ether and the like. Examples of the amide solvents include N, N-dimethylformamide, N, N-dimethyl tecetamide, N-methyl pyrrolidone, acetanilide and the like.
Examples of the amine solvents include ammonia, trimethylamine, triethylamine, n-butylamine, di-n-butylamine, tri-n-butylamine, aniline, N-methylaniline, N, N-dimethylaniline and pyridine.
These can be used alone or in combination of two or more solvents. Among these, ethanol, n-propanol, isopropanol and tetrahydrofuran are particularly preferable from the viewpoint of solubility in a resin and ease of purification.
　上記アセタール化は、酸触媒の存在下において行うことが好ましい。上記酸触媒は特に限定されず、硫酸、塩酸、硝酸、リン酸等の鉱酸や、ギ酸、酢酸、プロピオン酸等のカルボン酸や、メタンスルホン酸、エタンスルホン酸、ベンゼンスルホン酸、パラトルエンスルホン酸等のスルホン酸が挙げられる。これらの酸触媒は、単独で用いられてもよく、２種以上の化合物を併用してもよい。なかでも、塩酸、硝酸、硫酸が好ましく、塩酸が特に好ましい。 The acetalization is preferably performed in the presence of an acid catalyst. The above-mentioned acid catalyst is not particularly limited, and mineral acids such as sulfuric acid, hydrochloric acid, nitric acid and phosphoric acid, carboxylic acids such as formic acid, acetic acid and propionic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid and paratoluene sulfone And sulfonic acids such as acids. These acid catalysts may be used alone or in combination of two or more. Among them, hydrochloric acid, nitric acid and sulfuric acid are preferable, and hydrochloric acid is particularly preferable.
　本発明の幹細胞足場材料を使用することで、上記幹細胞種は特に問わないが、幹細胞足場材料として使用することができる。なかでも、多能性幹細胞、特にｉＰＳ細胞の培養に用いられることが好ましい。フィーダー細胞や接着タンパク質を含まない無血清培地培養において、幹細胞播種後の初期定着率が向上し、幹細胞の培養を好適に行うことができる。 The above-mentioned stem cell type is not particularly limited by using the stem cell scaffold material of the present invention, but it can be used as a stem cell scaffold material. Among them, it is preferable to be used for culturing pluripotent stem cells, particularly iPS cells. In a serum-free medium culture that does not contain feeder cells or adhesion proteins, the initial retention rate after seeding with stem cells is improved, and stem cells can be cultured preferably.
［幹細胞の培養方法］
　上述の幹細胞培養用足場材料によれば、様々な幹細胞を培養することができるが、その特性を考慮すると、幹細胞のなかでも多能性幹細胞の培養に用いることが好ましい。一般的に、多能性幹細胞は播種後の培養の定着率が低いとされているが、上述の幹細胞培養用足場材料は、培養培地の水分によって膨潤し難く、適度な親水性と強度を維持できるので、多能性幹細胞の播種後の定着率が向上するからである。 [Method for culturing stem cells]
According to the scaffold material for stem cell culture described above, various stem cells can be cultured, but in view of the characteristics, it is preferable to use for culturing pluripotent stem cells among stem cells. Generally, pluripotent stem cells are considered to have a low establishment rate in culture after seeding, but the above scaffold material for stem cell culture is not easily swelled by the water of the culture medium, and maintains adequate hydrophilicity and strength This is because the establishment rate after seeding of pluripotent stem cells is improved.
　幹細胞培養用足場材料は、幹細胞の培養において、平面培養（二次元培養方法）に用いることの他に、生体内により近い状態、例えば多孔質膜やハイドロゲルなどの基材上での幹細胞の培養（三次元培養方法）に用いることができる。細胞培養用足場材料をバイオリアクター等に用いることにより、効率良く幹細胞を増殖させることができるからである。
　細胞培養用足場材料は、適度な親水性と強度を備えることから、二次元培養方法に用いられることが好ましい。 The scaffold material for stem cell culture is, in addition to use for planar culture (two-dimensional culture method) in stem cell culture, a state closer to in vivo, for example, culture of stem cells on a substrate such as porous membrane or hydrogel It can be used for (three-dimensional culture method). This is because stem cells can be efficiently propagated by using a scaffold for cell culture for a bioreactor or the like.
The scaffold material for cell culture is preferably used in a two-dimensional culture method because it has appropriate hydrophilicity and strength.
　平面培養（二次元培養方法）用容器としては、形状や大きさは特に限定されないが、１つまたは複数のウェル（穴）を備える細胞培養用テストプレートや、細胞培養用フラスコ等が挙げられる。上記マイクロプレートのウェルの数は限定されないが、例えば、２、４、６、１２、２４、４８、９６、３８４等が挙げられる。上記ウェルの形状は特に限定されないが、真円、楕円、三角形、正方形、長方形、五角形等が挙げられる。上記ウェル底面の形状は特に限定されないが、平底、丸底、凹凸等が挙げられる。
　１つまたは複数のウェル（穴）を備える細胞培養用テストプレートや、細胞培養用フラスコの材質は特に限定されないが、高分子樹脂や金属、無機材料が挙げられる。上記高分子樹脂としては、ポリスチレン、ポリエチレン、ポリプロピレン、ポリカーボネート、ポリエステル、ポリイソプレン、シクロオレフィンポリマー、ポリイミド、ポリアミド、ポリアミドイミド、（メタ）アクリル樹脂、エポキシ樹脂、シリコーン等が挙げられる。金属としては、ステンレス、銅、鉄、ニッケル、アルミ、チタン、金、銀、白金等が挙げられる。無機材料としては、酸化ケイ素（ガラス）、酸化アルミ、酸化チタン、酸化ジルコニウム、酸化鉄、窒化ケイ素等が挙げられる。 The shape and size of the container for planar culture (two-dimensional culture method) are not particularly limited, and examples thereof include a cell culture test plate provided with one or more wells and a cell culture flask. Although the number of wells of the above-mentioned microplate is not limited, for example, 2, 4, 6, 12, 24, 48, 96, 384 and the like can be mentioned. The shape of the well is not particularly limited, and examples thereof include a perfect circle, an ellipse, a triangle, a square, a rectangle, a pentagon, and the like. The shape of the bottom of the well is not particularly limited, and examples thereof include a flat bottom, a round bottom, and irregularities.
The material of the cell culture test plate provided with one or more wells and the cell culture flask is not particularly limited, and examples thereof include polymer resins, metals, and inorganic materials. Examples of the polymer resin include polystyrene, polyethylene, polypropylene, polycarbonate, polyester, polyisoprene, cycloolefin polymer, polyimide, polyamide, polyamide imide, (meth) acrylic resin, epoxy resin, silicone and the like. Examples of the metal include stainless steel, copper, iron, nickel, aluminum, titanium, gold, silver, platinum and the like. Examples of the inorganic material include silicon oxide (glass), aluminum oxide, titanium oxide, zirconium oxide, iron oxide, silicon nitride and the like.
　上述の他にも、細胞培養用足場材料は、幹細胞を培地中で自由に浮遊させて成長させる浮遊培養方法に用いることができる。 In addition to the above, the scaffold for cell culture can be used in a suspension culture method in which stem cells are freely suspended and grown in a culture medium.
［多能性幹細胞の培養方法］
　多能性幹細胞の培養方法においては、合成樹脂を含有する幹細胞培養用足場材料上に細胞塊を播種することが好ましい。
　細胞塊は、コンフルエントになった培養容器に細胞剥離剤を添加し、ピペッティングにより均一に破砕処理することで得ることが出来る。細胞剥離剤としては、特に限定されないが、エチレンジアミン／リン酸緩衝溶液が好ましい。細胞塊の大きさは５０～２００μｍであることが好ましい。 [Method of culturing pluripotent stem cells]
In the method for culturing pluripotent stem cells, it is preferable to seed the cell mass on a scaffold for stem cell culture containing a synthetic resin.
A cell mass can be obtained by adding a cell release agent to a culture vessel that has become confluent and disrupting the cells uniformly by pipetting. The cell release agent is not particularly limited, but is preferably an ethylenediamine / phosphate buffer solution. The size of the cell mass is preferably 50 to 200 μm.
［その他の実施の形態］
　本発明は、上述の幹細胞培養用足場材料の他にも、その他の実施の形態として、幹細胞培養用足場材料を用いた発明が提供される。
　例えば、上述の幹細胞培養用足場材料と、多糖類と、を含有する幹細胞培養用担体（媒体）が提供される。多糖類としては、特に制限なく様々な多糖類を用いることができる。なかでも水溶性多糖類が好ましい。 [Other Embodiments]
The present invention provides an invention using a scaffold material for stem cell culture as another embodiment in addition to the scaffold material for stem cell culture described above.
For example, there is provided a stem cell culture carrier (medium) containing the above-described stem cell culture scaffold material and a polysaccharide. As polysaccharides, various polysaccharides can be used without particular limitation. Among them, water-soluble polysaccharides are preferred.
　細胞の培養領域の少なくとも一部に樹脂膜を備える幹細胞培養用容器であって、樹脂膜として、上述の幹細胞培養用足場材料が用いられた幹細胞培養用容器が提供される。容器としては、細胞の培養領域の少なくとも一部に樹脂膜を備えるものであれば、特に制限はなく、様々な容器を用いることができる。容器としては、上述の平面培養用容器や、バイオリアクター等を用いることができる。 A container for stem cell culture comprising a resin film in at least a part of a cell culture region, wherein the stem cell culture container using the scaffold material for stem cell culture described above as the resin film is provided. The container is not particularly limited as long as it has a resin film on at least a part of the cell culture area, and various containers can be used. As a container, the container for flat culture mentioned above, a bioreactor, etc. can be used.
　その他にも、幹細胞培養用足場材料を備える幹細胞培養用繊維が提供される。この場合、幹細胞培養用足場材料は、繊維上に塗布されていることが好ましい。また幹細胞培養用足場材料は、繊維中に含漬されたり、練り込まれている形態であってもよい。幹細胞培養用繊維は、フラスコなどの平面構造には接着しにくいが、線維（fibril）状構造などの立体構造には接着しやすい幹細胞の三次元培養方法に適している。幹細胞のなかでも、特に脂肪幹細胞の培養に適している。 In addition, a fiber for stem cell culture comprising a scaffold material for stem cell culture is provided. In this case, the scaffold material for stem cell culture is preferably coated on a fiber. In addition, the scaffold material for stem cell culture may be in the form of being impregnated or kneaded into fibers. The stem cell culture fiber is difficult to adhere to a planar structure such as a flask, but is suitable for a three-dimensional culture method of stem cells that easily adheres to a three-dimensional structure such as a fibril-like structure. Among stem cells, it is particularly suitable for culturing adipose stem cells.
　上記幹細胞培養用足場材料は架橋されていても良い。架橋されることで水膨潤性を抑制し、好適に強度を上げることができるからである。幹細胞培養用足場材料に更に架橋剤を加えて架橋させてもよい。
　架橋剤としては特に限定されないが、ポリアルコールやポリカルボン酸、ヒドロキシカルボン酸、金属石鹸、多糖類等が挙げられる。
　ポリアルコールとしては特に限定されないが、エチレングリコール、プロピレングリコール、ブタンジオール、ペンタンジオール、ヘキサンジオール、ヘプタンジオール、オクタンジオール、ノナンジオール、デカンジオール、ドデカンジオール、ウンデカンジオール、ジエチレングリコール、トリエチレングリコール、テトラエチレングリコール、ポリエチレングリコール、カテコール、ピロガロール、ジボロン酸、メチレンジボロン酸、エチレンジボロン酸、プロピレンジボロン酸、フェニレンジボロン酸、ビフェニルジボロン酸、ビスフェノール誘導体等が挙げられる。
　ポリカルボン酸としては特に限定されないが、シュウ酸、マロン酸、コハク酸、グルタル酸、アジピン酸、ピメリン酸、スベリン酸、アゼライン酸、セバシン酸、フタル酸、ポリ（メタ）アクリル酸等が挙げられる。
　ヒドロキシカルボン酸としては特に限定されないが、グリコール酸、乳酸、タルトロン酸、グリセリン酸、ヒドロキシ酪酸、リンゴ酸、酒石酸、シトマル酸、クエン酸、イソクエン酸、ロイシン酸、メバロン酸、パントイン酸、リシノール酸、リシネライジン酸、セレブロン酸、キナ酸、シキミ酸、ヒドロキシ安息香酸、サリチル酸、クレオソート酸、バニリン酸、シリング酸、ピロカテク酸、レソルシル酸、プロトカテク酸、ゲンチジン酸、オルセリン酸、没食子酸、マンデル酸、ベンジル酸、アトロラクチン酸、メリロト酸、フロレト酸、クマル酸、ウンベル酸、コーヒー酸、フェルラ酸、シナピン酸、ヒドロキシステアリン酸等が挙げられる。
　金属石鹸としては特に限定されないが、ステアリン酸、ラウリン酸、リシノール酸、オクチル酸などの脂肪酸と、リチウム、ナトリウム、マグネシウム、カルシウム、バリウム、亜鉛、アルミニウムなどの金属の塩が挙げられる。
多糖類としては特に限定されないが、ペクチン、グアーガム、キサンタンガム、タマリンドガム、カラギーナン、プロピレングリコール、カルボキシメチルセルロース、アミロース、アミロペクチン、グリコーゲン、セルロース、キチン、アガロース、カラギーナン、ヘパリン、ヒアルロン酸、キシログルカン、グルコマンナン酸等が挙げられる。 The scaffold material for stem cell culture may be crosslinked. It is because water swelling property can be suppressed and strength can be raised suitably by being bridged. A crosslinking agent may be further added to the scaffold material for stem cell culture to cause crosslinking.
The crosslinking agent is not particularly limited, and examples thereof include polyalcohols, polycarboxylic acids, hydroxycarboxylic acids, metal soaps, polysaccharides and the like.
The polyalcohol is not particularly limited, but ethylene glycol, propylene glycol, butanediol, pentanediol, hexanediol, heptanediol, octanediol, nonanediol, decanediol, dodecanediol, undecanediol, undecanediol, diethylene glycol, triethylene glycol, tetraethylene Examples thereof include glycol, polyethylene glycol, catechol, pyrogallol, diboronic acid, methylene diboronic acid, ethylene diboronic acid, propylene diboronic acid, phenylene diboronic acid, biphenyl diboronic acid, and bisphenol derivatives.
The polycarboxylic acid is not particularly limited, and oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, phthalic acid, poly (meth) acrylic acid and the like can be mentioned. .
The hydroxycarboxylic acid is not particularly limited, but glycolic acid, lactic acid, thalthronic acid, glyceric acid, hydroxybutyric acid, malic acid, tartaric acid, tartaric acid, citric acid, isocitric acid, leucine acid, mevalonic acid, pantoic acid, ricinoleic acid, Ricinereidiic acid, cerebronic acid, quinic acid, shikimic acid, hydroxybenzoic acid, salicylic acid, creosote acid, vanillic acid, silicic acid, pyrocatechic acid, resorcylic acid, protocatechuic acid, gentisic acid, orsellinic acid, gallic acid, mandelic acid, benzyl Acids, atrolactic acid, melilotonic acid, phlorettic acid, coumaric acid, umbellic acid, caffeic acid, ferulic acid, sinapinic acid, hydroxystearic acid and the like can be mentioned.
The metal soap is not particularly limited, and examples thereof include fatty acids such as stearic acid, lauric acid, ricinoleic acid and octylic acid, and salts of metals such as lithium, sodium, magnesium, calcium, barium, zinc and aluminum.
The polysaccharides are not particularly limited, but pectin, guar gum, xanthan gum, tamarind gum, carrageenan, propylene glycol, carboxymethylcellulose, amylose, amylopectin, glycogen, cellulose, chitin, agarose, carrageenan, heparin, hyaluronic acid, xyloglucan, glucomannan An acid etc. are mentioned.
　以下に、実施例及び比較例を挙げて本発明を説明するが本発明は以下の実施例に限定解釈されることはない。なお、得られた合成樹脂、変性ポリビニルアセタール樹脂における構成単位、例えば、アミン構造を有する構成単位の含有量（モル％）、イミン構造を有する構成単位の含有量（モル％）、アミド構造を有する構成単位の含有量（モル％）、アセタール化度（モル％）、アセチル基量（モル％）、水酸基量（モル％）、（メタ）アクリル酸エステル基量（モル％）は、合成樹脂をＤＭＳＯ－ｄ６（ジメチルスルホキサイド）に溶解し、１Ｈ-ＮＭＲ（核磁気共鳴スペクトル）を用いて測定した。 Hereinafter, the present invention will be described by way of examples and comparative examples, but the present invention is not limited to the following examples. The content of the structural unit in the obtained synthetic resin and modified polyvinyl acetal resin, for example, the structural unit having an amine structure (mol%), the content of the structural unit having an imine structure (mol%), and an amide structure The content (mol%) of the constituent units, the degree of acetalization (mol%), the amount of acetyl groups (mol%), the amount of hydroxyl groups (mol%), and the amount of (meth) acrylic acid ester groups (mol%) It was dissolved in DMSO-d6 (dimethyl sulfoxide) and measured using 1 H-NMR (nuclear magnetic resonance spectrum).
［実施例１］
（ポリビニルブチラールの調製）
　攪拌装置を備えた反応機に、イオン交換水２７００ｍＬ、平均重合度２５０、鹸化度９９モル％のポリビニルアルコールを３００ｇ投入し、攪拌しながら加熱溶解し、溶液を得た。次に、この溶液に触媒として３５重量％塩酸を、塩酸濃度が０．２重量％となるように添加し、温度を１５℃に調整した後、攪拌しながらｎ-ブチルアルデヒド(ｎ-ＢＡ)２２ｇを添加した。その後、ｎ-ブチルアルデヒド(ｎ-ＢＡ)１４８ｇを添加したところ、白色粒子状のポリビニルブチラールが析出した。析出してから１５分後に、３５重量％塩酸を、塩酸濃度が１．８重量％になるように添加し、５０℃に加熱し、５０℃で２時間熟成させた。次いで、溶液を冷却し、中和した後、ポリビニルブチラールを水洗し、乾燥させることにより、ポリビニルブチラールを得た。
　得られたポリビニルブチラールは、平均重合度２５０、水酸基量２８モル％、アセチル基量１モル％、アセタール化度７１モル％であった。 Example 1
(Preparation of polyvinyl butyral)
In a reactor equipped with a stirrer, 300 g of polyvinyl alcohol having 2700 mL of ion-exchanged water, an average polymerization degree of 250, and a saponification degree of 99 mol% was charged and dissolved with heating while stirring to obtain a solution. Next, 35 wt% hydrochloric acid as a catalyst is added to this solution so that the concentration of hydrochloric acid is 0.2 wt%, the temperature is adjusted to 15 ° C., and n-butyraldehyde (n-BA) is stirred. 22 g was added. Thereafter, 148 g of n-butyraldehyde (n-BA) was added to precipitate white particles of polyvinyl butyral. 15 minutes after precipitation, 35 wt% hydrochloric acid was added so that the concentration of hydrochloric acid was 1.8 wt%, heated to 50 ° C., and aged at 50 ° C. for 2 hours. Then, the solution was cooled and neutralized, and then polyvinyl butyral was washed with water and dried to obtain polyvinyl butyral.
The obtained polyvinyl butyral had an average degree of polymerization of 250, an amount of hydroxyl groups of 28 mol%, an amount of acetyl groups of 1 mol%, and an acetalization degree of 71 mol%.
（細胞培養用容器の調製）
　得られたポリビニルブチラール１ｇをブタノール１９ｇに溶解させることで、ポリビニルブチラール溶液を得た。得られたポリビニルブチラール溶液１５０μＬをφ２２ｍｍのカバーガラス（松浪社製、２２丸Ｎｏ．１をエアダスターで除塵して使用）上に吐出し、スピンコーターを用いて２０００ｒｐｍ、２０秒回転させて平滑な樹脂膜を得た。得られた上記樹脂膜をカバーガラスごとφ２２ｍｍのポリスチレンディッシュに配置することにより細胞培養用容器を得た。 (Preparation of container for cell culture)
A polyvinyl butyral solution was obtained by dissolving 1 g of the obtained polyvinyl butyral in 19 g of butanol. Discharge 150 μL of the obtained polyvinyl butyral solution onto a φ22 mm cover glass (Matsunami Co., Ltd., using No. 1 dust removed with an air duster) and spin it at 2000 rpm for 20 seconds using a spin coater to make it smooth A resin film was obtained. The obtained resin film was placed on a polystyrene dish with a diameter of 22 mm together with the cover glass to obtain a cell culture vessel.
（表面自由エネルギー）
　上記樹脂膜の表面自由エネルギーについて接触角計（協和界面化学社製、ＤＭｏー７０１）を用いて測定した。上記樹脂膜上に純水１μＬを着滴させ、３０秒後の液滴像を撮影することで純水の接触角を得た。また、上記樹脂膜上にジヨードメタン１μＬを着滴させ、３０秒後の液滴像を撮影することでジヨードメタンの接触角を得た。得られた上記接触角をＫａｅｌｂｌｅ－Ｕｙ理論を用いて表面自由エネルギーγ、分散成分γｄ、双極子成分γｐを導出した。 (Surface free energy)
The surface free energy of the resin film was measured using a contact angle meter (DMo-701, manufactured by Kyowa Interface Chemical Co., Ltd.). A contact angle of pure water was obtained by depositing 1 μl of pure water on the resin film and photographing a droplet image after 30 seconds. Further, 1 μL of diiodomethane was deposited on the resin film, and a droplet image after 30 seconds was taken to obtain a contact angle of diiodomethane. The surface free energy γ, the dispersion component γ d , and the dipole component γ p were derived from the obtained contact angles using the Kaelble-Uy theory.
　以下の条件において樹脂膜を備える細胞培養用容器について試験を行った。
（細胞培養試験の方法）
　得られた細胞培養用容器にリン酸緩衝生理食塩水１ｍＬを加えて３７℃のインキュベーター内で１時間静置した。ディッシュ内のリン酸緩衝生理食塩水を除いた後、ｈ－ｉＰＳ細胞２５３Ｇ１を１．５×１０４を播種し、培地ＴｅＳＲ Ｅ８（ＳＴＥＭ　ＣＥＬＬ社製）１ｍＬおよび、ＲＯＣＫ－Ｉｎｈｉｂｉｔｏｒ（Ｙ２７６３２）１０μＭ存在下、３７℃、ＣＯ２濃度５％のインキュベーター内で培養を行った。２４時間毎に培地を７５０μＬ除き、新たなＴｅＳＲ Ｅ８ ２５０μＬを加え、ＲＯＣＫ－Ｉｎｈｉｂｉｔｏｒ（Ｙ２７６３２）１０μＭに調整することで培地交換を行った。 Tests were conducted on a cell culture vessel provided with a resin film under the following conditions.
(Method of cell culture test)
1 mL of phosphate buffered saline was added to the obtained cell culture vessel, and the cells were allowed to stand in a 37 ° C. incubator for 1 hour. After removing phosphate buffered saline in the dish, 1.5 × 10 4 h-iPS cells 253G1 are seeded, 1 mL of medium TeSR E8 (STEM CELL) and 10 μM of ROCK-Inhibitor (Y27632) are present. Culturing was carried out at 37 ° C. in a 5% CO 2 incubator. The medium was changed by removing 750 μl of the medium every 24 hours, adding 250 μl of fresh TeSR E8, and adjusting to 10 μM of ROCK-Inhibitor (Y27632).
（細胞塊培養試験の方法）
　得られた細胞培養用容器にリン酸緩衝生理食塩水１ｍＬを加えて３７℃のインキュベーター内で１時間静置後、培養容器内のリン酸緩衝生理食塩水を除いた。３５ｍｍディッシュにコンフルエント状態になったｈ－ｉＰＳ細胞２５２Ｇ１のコロニーを加え、次に１ｍＬの０．５ｍＭエチレンジアミン／リン酸緩衝溶液を加え、室温で２分静置した。その後、エチレンジアミン/リン酸緩衝溶液を除き、１ｍＬのＴｅＳＲＥ８培地でピペッティングにより５０～２００μｍに砕かれた細胞塊を１．０×１０５を培養容器に播種し、培地ＴｅＳＲ Ｅ８（ＳＴＥＭ　ＣＥＬＬ社製）１ｍＬおよび、ＲＯＣＫ－Ｉｎｈｉｂｉｔｏｒ（Ｙ２７６３２）１０μＭ存在下、３７℃、ＣＯ２濃度５％のインキュベーター内で培養を行った。２４時間毎に培地を７５０μＬ除き、新たなＴｅＳＲ Ｅ８を２５０μＬ加えることで培地交換を行った。 (Method of cell mass culture test)
1 mL of phosphate buffered saline was added to the obtained cell culture vessel, and after standing for 1 hour in a 37 ° C. incubator, the phosphate buffered saline in the culture vessel was removed. Confluent colonies of h-iPS cells 252G1 were added to a 35 mm dish, then 1 mL of a 0.5 mM ethylenediamine / phosphate buffer solution was added and allowed to stand at room temperature for 2 minutes. Thereafter, the ethylenediamine / phosphate buffer solution is removed, and the cell mass crushed to 50 to 200 μm by pipetting with 1 mL of TeSRE 8 medium is inoculated with 1.0 × 10 5 cells into a culture vessel, and medium TeSR E8 (STEM CELL Co., Ltd.) 2.) Cultivation was carried out in the presence of 1 mL and 10 μM of ROCK-Inhibitor (Y27632) at 37 ° C. in a 5% CO 2 incubator. The medium was changed by removing 750 μl of the medium every 24 hours and adding 250 μl of fresh TeSR E8.
（培養評価の方法）
（１）初期接着性
　細胞培養試験において、細胞播種後２４時間後の細胞像を位相差顕微鏡１０×１０倍の位相差顕微鏡（オリンパス社製、ＩＸ７３）を用いて取得した。その際、培養容器内の最も平均的な接着形態を示す視野の画像の取得を行った。得られた画像を図４の見本１～見本１０と照らし合わせて、接着細胞数と接着細胞形態を加味して初期接着性の評価を行った。なお、図４において、見本１から見本８に向かうほど細胞が増加していることが示されている。また見本８から見本１０に向かうほど、細胞の仮足が伸長し、より良好な接着状態にあることが示されている。得られた結果を図５、６にまとめて示す。
（２）細胞増殖性
　細胞培養試験において、細胞播種後５日経過後の細胞像を位相差顕微鏡１０×４倍の位相差顕微鏡（オリンパス社製、ＩＸ７３）を用いて取得した。その際、培養容器内の最も平均的な接着形態を示す視野の画像の取得を行った。得られた画像を図７の見本１～見本１０と照らし合わせることで細胞増殖性の評価を行った。図７において、細胞増殖によりコロニーが成長するほど高評価とした。なお、コロニーは横方向（画面の縦横方向）に成長し過ぎると縦方向（画面の手前側方向）に積み重なり始めるため、光の透過性が低くなる傾向がある。得られた結果を図８、９にまとめて示す。
（３）接着維持性
　細胞塊培養試験において、細胞塊の接着維持可能時間を以下の基準に従って評価した。
　０：培地交換後３０分未満で全ての細胞が剥離した。
　１：培地交換後３０分以上接着維持したが、１時間未満で全ての細胞が剥離した。
　２：培地交換後１時間以上接着維持したが、２４時間未満で全ての細胞が剥離した。
　３：培地交換後２４時間以上接着維持した。
　得られた細胞塊はアルカリフォスファターゼ（ＡＬＰ）染色試験により未分化性が保たれていることを確認した。 (Method of culture evaluation)
(1) Initial Adhesion In a cell culture test, a cell image 24 hours after cell seeding was obtained using a phase contrast microscope 10 × 10 × phase contrast microscope (IX 73, manufactured by Olympus Corporation). At that time, an image of a field of view showing the most average adhesion form in the culture vessel was obtained. The obtained image was compared with sample 1 to sample 10 in FIG. 4 to evaluate the initial adhesiveness by considering the number of adherent cells and the form of adherent cells. In FIG. 4, it is shown that the cells increase from sample 1 to sample 8. It is also shown that, from sample 8 to sample 10, the pseudopods of cells are elongated and in a better adhesion state. The obtained results are summarized in FIGS.
(2) Cell Proliferation In the cell culture test, cell images after 5 days after cell seeding were obtained using a phase contrast microscope 10 × 4 × phase contrast microscope (IX 73, manufactured by Olympus Corporation). At that time, an image of a field of view showing the most average adhesion form in the culture vessel was obtained. The obtained images were compared with Samples 1 to 10 in FIG. 7 to evaluate cell proliferation. In FIG. 7, it was evaluated highly as colonies grew by cell growth. In addition, since the colonies start to be stacked in the vertical direction (front side direction of the screen) when the colonies grow too much in the horizontal direction (vertical and horizontal direction of the screen), light transmittance tends to be low. The obtained results are summarized in FIGS.
(3) Adhesion maintenance property In the cell mass culture test, adhesion maintenance possible time of cell mass was evaluated according to the following criteria.
0: All cells detached in less than 30 minutes after medium replacement.
1: Adhesion was maintained for 30 minutes or more after medium replacement, but all cells were detached in less than 1 hour.
2: Adhesion was maintained for 1 hour or more after medium replacement, but all cells were detached in less than 24 hours.
3: Adhesion was maintained for 24 hours or more after medium replacement.
The obtained cell mass was confirmed to be undifferentiated by alkaline phosphatase (ALP) staining test.
［実施例２］
　平均重合度８５０、鹸化度９９モル％のポリビニルアルコールを使用したこと以外は、実施例１同様にして試験を行った。 Example 2
The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average polymerization degree of 850 and a saponification degree of 99 mol% was used.
［実施例３］
　平均重合度１７００、鹸化度９９モル％のポリビニルアルコールを使用したこと以外は、実施例１同様にして試験を行った。 [Example 3]
The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average degree of polymerization of 1700 and a degree of saponification of 99 mol% was used.
［実施例４］
　平均重合度２４００、鹸化度９９モル％のポリビニルアルコールを使用したこと、および、ｎ-ブチルアルデヒド(ｎ-ＢＡ)の代わりに、アセトアルデヒドを使用すること以外は、実施例１同様にして試験を行った。 Example 4
The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average degree of polymerization of 2400 and a degree of saponification of 99 mol% was used, and that acetaldehyde was used instead of n-butyraldehyde (n-BA). The
［実施例５］
　平均重合度８５０、鹸化度９８モル％、エチレン変性度４モル％のポリビニルアルコールを使用したこと以外は、実施例１同様にして試験を行った。 [Example 5]
The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average polymerization degree of 850, a saponification degree of 98 mol% and an ethylene modification degree of 4 mol% was used.
［実施例６］
　平均重合度２５０、鹸化度９９モル％、上記式（３）に示すアミノ基を有する構成単位を２モル％含有するポリビニルアルコールを使用したこと以外は、実施例１同様にして試験を行った。 [Example 6]
The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average degree of polymerization of 250, a degree of saponification of 99 mol%, and 2 mol% of a constituent unit having an amino group shown in the above formula (3) was used.
［実施例７］
　平均重合度１６００、鹸化度９９モル％、上記式（３）に示すアミノ基を有する構成単位を２モル％含有するポリビニルアルコールを使用したこと以外は、実施例１同様にして試験を行った。 [Example 7]
The test was conducted in the same manner as in Example 1 except that a polyvinyl alcohol having an average polymerization degree of 1600, a saponification degree of 99 mol%, and a structural unit having an amino group shown in the above formula (3) was used.
［実施例８］
　実施例１で得られた重合度約２５０のポリビニルアセタール１００重量部、および、Ｎ－ビニルピロリドン１重量部を５００重量部のテトラヒドロフランに溶解させてグラフト共重合体樹脂溶液を得た。得られた樹脂溶液にＩｒｇａｃｕｒｅ１８４（ＢＡＳＦ社製）０．０５重量部を溶解させ、ＰＥＴフィルム上に塗布した。塗布物を２５℃にてアイグラフィックス社製、ＵＶコンベア装置「ＥＣＳ３０１Ｇ１」を用い、３６５ｎｍの波長の光を積算光量２０００ｍＪ／ｃｍ２で照射することで複合樹脂溶液を得た。得られた複合樹脂溶液を８０℃、３時間真空乾燥させることで複合樹脂を得た。得られた樹脂について、カラムとしてＷａｔｅｒｓ社製「２６９０　Ｓｅｐａｒａｔｉｏｎｓ　Ｍｏｄｅｌ」を用いて、ＧＰＣ法によってポリスチレン換算による重量平均分子量を測定したところ、約４万であった。得られた複合樹脂を３重量％ブタノール溶液に調整し、実施例１同様にして試験を行った。 [Example 8]
100 parts by weight of polyvinyl acetal having a degree of polymerization of about 250 obtained in Example 1 and 1 part by weight of N-vinylpyrrolidone were dissolved in 500 parts by weight of tetrahydrofuran to obtain a graft copolymer resin solution. In the obtained resin solution, 0.05 parts by weight of Irgacure 184 (manufactured by BASF Corporation) was dissolved and applied onto a PET film. A composite resin solution was obtained by irradiating a light having a wavelength of 365 nm with an integrated light quantity of 2000 mJ / cm 2 using a UV conveyor apparatus “ECS 301 G1” manufactured by Eye Graphics Co., Ltd. at 25 ° C. The resulting composite resin solution was vacuum dried at 80 ° C. for 3 hours to obtain a composite resin. It was about 40,000 when the weight average molecular weight by polystyrene conversion was measured by GPC method using "2690 Separations Model" by Waters company about the obtained resin as a column. The obtained composite resin was adjusted to a 3% by weight butanol solution, and tested in the same manner as in Example 1.
［実施例９］
　ポリビニルアセタール１００重量部に対してＮ－ビニルピロリドン１０重量部を添加したこと以外は、実施例８同様にして試験を行った。得られた樹脂の重量平均分子量は約６万であった。 [Example 9]
The test was conducted in the same manner as in Example 8 except that 10 parts by weight of N-vinylpyrrolidone was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 60,000.
［実施例１０］
　ポリビニルアセタール１００重量部に対してＮ－ビニルピロリドン３０重量部を添加したこと以外は、実施例８同様にして試験を行った。得られた樹脂の重量平均分子量は約５万であった。 [Example 10]
The test was conducted in the same manner as in Example 8 except that 30 parts by weight of N-vinylpyrrolidone was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 50,000.
［実施例１１］
　ポリビニルアセタール１００重量部に対してテトラヒドロフルフリルアクリレート５重量部を添加したこと以外は、実施例８同様にして試験を行った。得られた樹脂の重量平均分子量は約６万であった。 [Example 11]
The test was conducted in the same manner as in Example 8 except that 5 parts by weight of tetrahydrofurfuryl acrylate was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 60,000.
［実施例１２］
　ポリビニルアセタール１００重量部に対してメトキシエチルアクリレート５重量部を添加したこと以外は、実施例８同様にして試験を行った。得られた樹脂の重量平均分子量は約７万であった。 [Example 12]
The test was conducted in the same manner as in Example 8 except that 5 parts by weight of methoxyethyl acrylate was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 70,000.
［実施例１３］
　ポリビニルアセタール１００重量部に対してブチルメタクリレート５重量部を添加したこと以外は、実施例８同様にして試験を行った。得られた樹脂の重量平均分子量は約６万。 [Example 13]
The test was conducted in the same manner as in Example 8 except that 5 parts by weight of butyl methacrylate was added to 100 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin is about 60,000.
［実施例１４］
　Ｎ－イソプロピルアクリルアミド７５重量部、および、ブチルメタクリレート２５重量部をテトラヒドロフラン３００重量部に溶解させてアクリルモノマー溶液を得た。得られたアクリルモノマー溶液にＩｒｇａｃｕｒｅ１８４（ＢＡＳＦ社製）２重量部を溶解させ、ＰＥＴフィル上に塗布した。塗布物を２５℃にてアイグラフィックス社製、ＵＶコンベア装置「ＥＣＳ３０１Ｇ１」を用い、３６５ｎｍの波長の光を積算光量２０００ｍＪ／ｃｍ２で照射することでアクリル樹脂溶液を得た。得られたアクリル樹脂溶液を８０℃、３時間真空乾燥させることでアクリル樹脂を得た。得られたアクリル樹脂を３重量％ブタノール溶液に調整し、実施例１同様にして試験を行った。得られたアクリル樹脂の重量平均分子量は約１０万であった。 Example 14
An acrylic monomer solution was obtained by dissolving 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate in 300 parts by weight of tetrahydrofuran. Two parts by weight of Irgacure 184 (manufactured by BASF Corp.) was dissolved in the obtained acrylic monomer solution, and the solution was coated on a PET film. An acrylic resin solution was obtained by irradiating a light having a wavelength of 365 nm with an integrated light quantity of 2000 mJ / cm 2 using a UV conveyor apparatus “ECS 301 G1” manufactured by Eye Graphics Co., Ltd. at 25 ° C. The obtained acrylic resin solution was vacuum dried at 80 ° C. for 3 hours to obtain an acrylic resin. The resulting acrylic resin was adjusted to a 3% by weight butanol solution, and tested in the same manner as in Example 1. The weight average molecular weight of the obtained acrylic resin was about 100,000.
［実施例１５］
　Ｎ－イソプロピルアクリルアミド７５重量部、および、ブチルメタクリレート２５重量部に代えて、メトキシエチルアクリレート９０重量部、および、ブチルメタクリレート１０重量部を使用したこと以外は実施例１４と同様にして、アクリル樹脂を得た。得られたアクリル樹脂を３重量％ブタノール溶液に調整し、実施例１同様にして試験を行った。得られたアクリル樹脂の重量平均分子量は約８万であった。 [Example 15]
An acrylic resin was prepared in the same manner as in Example 14 except that 90 parts by weight of methoxyethyl acrylate and 10 parts by weight of butyl methacrylate were used instead of 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate. Obtained. The resulting acrylic resin was adjusted to a 3% by weight butanol solution, and tested in the same manner as in Example 1. The weight average molecular weight of the obtained acrylic resin was about 80,000.
［実施例１６］
　Ｎ－イソプロピルアクリルアミド７５重量部、および、ブチルメタクリレート２５重量部に代えて、メトキシエチルアクリレート７５重量部、および、ブチルメタクリレート２５重量部を使用したこと以外は実施例１４と同様にして、アクリル樹脂を得た。得られたアクリル樹脂を３重量％ブタノール溶液に調整し、実施例１同様にして試験を行った。得られた樹脂の重量平均分子量は約９万であった。 [Example 16]
An acrylic resin was prepared in the same manner as in Example 14 except that 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate were used, and 75 parts by weight of methoxyethyl acrylate and 25 parts by weight of butyl methacrylate were used. Obtained. The resulting acrylic resin was adjusted to a 3% by weight butanol solution, and tested in the same manner as in Example 1. The weight average molecular weight of the obtained resin was about 90,000.
［実施例１７］
　Ｎ－イソプロピルアクリルアミド７５重量部、および、ブチルメタクリレート２５重量部に代えて、ブチルメタクリレート２重量部、および、エチルアクリレート９８重量部を使用したこと以外は実施例１４と同様にして、アクリル樹脂を得た。得られたアクリル樹脂を３重量％ブタノール溶液に調整し、実施例１同様にして試験を行った。得られたアクリル樹脂の重量平均分子量は約８万であった。 [Example 17]
An acrylic resin is obtained in the same manner as in Example 14 except that 2 parts by weight of butyl methacrylate and 98 parts by weight of ethyl acrylate are used instead of 75 parts by weight of N-isopropylacrylamide and 25 parts by weight of butyl methacrylate. The The resulting acrylic resin was adjusted to a 3% by weight butanol solution, and tested in the same manner as in Example 1. The weight average molecular weight of the obtained acrylic resin was about 80,000.
［比較例１］
　足場材料を用いず、ポリスチレンディッシュのみで実施例１同様にて試験を行った。 Comparative Example 1
The test was performed in the same manner as in Example 1 using only a polystyrene dish without using a scaffold material.
［比較例２］
　２回目のｎ-ブチルアルデヒド(ｎ-ＢＡ)の添加量を１４８ｇから８９ｇに変更したこと以外は、実施例１同様にして試験を行った。 Comparative Example 2
The test was performed in the same manner as in Example 1 except that the second addition amount of n-butyraldehyde (n-BA) was changed from 148 g to 89 g.
［比較例３］
　合成樹脂として平均重合度１０００、鹸化度９８モル％のポリビニルアルコールを使用したこと以外は、実施例１同様にして試験を行った。 Comparative Example 3
The test was conducted in the same manner as in Example 1 except that polyvinyl alcohol having an average polymerization degree of 1000 and a saponification degree of 98 mol% was used as the synthetic resin.
［比較例４］
　Ｎ－イソプロピルアクリルアミド１００重量部、酢酸エチル７５重量部、アゾビスイソブチロニトリル０．５重量部を混合し、窒素雰囲気下、６５℃で８時間重合を行うことでポリアクリルアミド樹脂を得た。得られた樹脂について、カラムとしてＷａｔｅｒｓ社製「２６９０　Ｓｅｐａｒａｔｉｏｎｓ　Ｍｏｄｅｌ」を用いて、ＧＰＣ法によってポリスチレン換算による重量平均分子量を測定したところ、約９万（重合度約８００）であった。その他の操作は実施例１同様にして試験を行った。 Comparative Example 4
A polyacrylamide resin was obtained by mixing 100 parts by weight of N-isopropyl acrylamide, 75 parts by weight of ethyl acetate and 0.5 parts by weight of azobisisobutyronitrile, and performing polymerization at 65 ° C. for 8 hours in a nitrogen atmosphere. It was about 90,000 (degree of polymerization about 800) when the weight average molecular weight by polystyrene conversion was measured by GPC method using "2690 Separations Model" by Waters company about the obtained resin as a column. The other operations were tested in the same manner as in Example 1.
［比較例５］
　Ｎ－イソプロピルアクリルアミド１００重量部に代えてエチルアクリレート１００重量部を使用したこと以外は比較例４と同様にして試験を行った。 Comparative Example 5
The test was conducted in the same manner as Comparative Example 4 except that 100 parts by weight of ethyl acrylate was used instead of 100 parts by weight of N-isopropylacrylamide.
［比較例６］
　Ｎ－イソプロピルアクリルアミド１００重量部に代えてブチルメタクリレート１００重量部を使用したこと以外は比較例４と同様にして試験を行った。得られた樹脂の重量平均分子量は約９万であった。 Comparative Example 6
The test was conducted in the same manner as Comparative Example 4 except that 100 parts by weight of butyl methacrylate was used instead of 100 parts by weight of N-isopropylacrylamide. The weight average molecular weight of the obtained resin was about 90,000.
［比較例７］
　ポリビニルアセタール３０重量部に対してＮ－ビニルピロリドン７０重量部を添加したこと以外は、実施例８同様にして試験を行った。得られた樹脂の重量平均分子量は約９万であった。 Comparative Example 7
The test was conducted in the same manner as in Example 8 except that 70 parts by weight of N-vinylpyrrolidone was added to 30 parts by weight of polyvinyl acetal. The weight average molecular weight of the obtained resin was about 90,000.
　得られた結果をまとめて表１、表２に示す。図５、図６に播種後２４時間後の細胞の位相差顕微鏡写真を示す。図８、図９に播種後５日後の細胞の位相差顕微鏡写真を示す。なお、いずれの実施例及び比較例においても分化した細胞は観察されなかった。 
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
 The obtained results are summarized in Tables 1 and 2. The phase contrast micrographs of the cells after 24 hours after seeding are shown in FIG. 5 and FIG. The phase contrast micrograph of the cell 5 days after seeding is shown in FIG. 8 and FIG. In addition, differentiated cells were not observed in any of the examples and comparative examples.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008

Claims (12)

  1. 　表面自由エネルギーの分散成分γｄが２４．５以上４５．０未満であり、
    　表面自由エネルギーの双極子成分γｐが１．０以上２０．０未満である幹細胞培養用足場材料。     The dispersion component γ d of surface free energy is 24.5 or more and less than 45.0,
    A scaffold material for stem cell culture, wherein a dipole component γ p of surface free energy is 1.0 or more and less than 20.0.
  2. 　前記幹細胞培養用足場材料が合成樹脂を含む、請求項１記載の幹細胞培養用足場材料。 The scaffold material for stem cell culture according to claim 1, wherein the scaffold material for stem cell culture comprises a synthetic resin.
  3. 　前記合成樹脂がポリビニルアセタール骨格、ポリ（メタ）アクリル酸エステル骨格の少なくともいずれか一方を含む請求項２記載の幹細胞培養用足場材料。 The scaffold material for stem cell culture according to claim 2, wherein the synthetic resin contains at least one of a polyvinyl acetal skeleton and a poly (meth) acrylic acid ester skeleton.
  4. 　前記合成樹脂がポリビニルアセタール樹脂である請求項２記載の幹細胞培養用足場材料。 The scaffold material for stem cell culture according to claim 2, wherein the synthetic resin is a polyvinyl acetal resin.
  5. 　合成樹脂を含有する幹細胞培養用足場材料であって、
    　前記合成樹脂がポリビニルアセタール樹脂を含み、
    　前記ポリビニルアセタール樹脂のアセタール化度が６０モル％よりも高い幹細胞培養用足場材料。     A scaffold material for stem cell culture, comprising a synthetic resin, wherein
    The synthetic resin comprises polyvinyl acetal resin,
    The scaffold material for stem cell culture whose degree of acetalization of the said polyvinyl acetal resin is higher than 60 mol%.
  6. 　前記ポリビニルアセタール樹脂は、アミン構造を有する構成単位、イミン構造を有する構成単位、及びアミド構造を有する構成単位からなる群から選択される少なくとも一種を構成単位として含む、請求項４または５記載の幹細胞培養用足場材料。 The stem cell according to claim 4 or 5, wherein the polyvinyl acetal resin contains at least one selected from the group consisting of a constituent unit having an amine structure, a constituent unit having an imine structure, and a constituent unit having an amide structure as a constituent unit. Scaffold material for culture.
  7. 　前記ポリビニルアセタール樹脂は、アミン構造を有する構成単位、イミン構造を有する構成単位、及びアミド構造を有する構成単位の合計の含有量が、０．１モル％以上３０モル％以下である、請求項６記載の幹細胞培養用足場材料。 The polyvinyl acetal resin has a total content of a structural unit having an amine structure, a structural unit having an imine structure, and a structural unit having an amide structure is 0.1 mol% or more and 30 mol% or less. Scaffold material for stem cell culture as described.
  8. 　前記幹細胞が多能性幹細胞である請求項１～７のいずれか１項記載の幹細胞培養用足場材料。 The scaffold material for stem cell culture according to any one of claims 1 to 7, wherein the stem cells are pluripotent stem cells.
  9. 　細胞の培養領域の少なくとも一部に請求項１～８のいずれか１項記載の幹細胞培養用足場材料を含む、幹細胞培養用容器。 A container for stem cell culture, comprising the scaffold for stem cell culture according to any one of claims 1 to 8 in at least a part of the culture area of cells.
  10. 　請求項１～８のいずれか１項記載の幹細胞培養用足場材料を含む、幹細胞培養用繊維。 A stem cell culture fiber comprising the scaffold material for stem cell culture according to any one of claims 1 to 8.
  11. 　請求項１～８のいずれか１項記載の幹細胞培養用足場材料を用いる幹細胞の培養方法。 A method for culturing stem cells using the scaffold for stem cell culture according to any one of claims 1 to 8.
  12. 　前記幹細胞培養用足場材料上に細胞塊を播種する工程を含む、請求項１１記載の幹細胞の培養方法。
          The method for culturing stem cells according to claim 11, comprising the step of seeding a cell mass on the scaffold for stem cell culture.
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